BACKGROUND::

Several techniques are used to measure infliximab (IFX) and anti-IFX antibodies (Abs) in Crohn's disease. The aim of this study was to compare different assays for this purpose.

METHODS::

Fluid-phase radioimmunoassay (RIA), solid-phase enzyme-linked immunosorbent assay (ELISA), reporter gene assay (RGA), and enzyme immunoassay (EIA; anti-IFX Ab only) were assessed. IFX was added to pooled serum from 13 patients with inactive Crohn's disease to yield concentrations of 0, 1, 3, and 9 µg/mL. Anti-IFX Abs were assessed in 6 patients.

RESULTS::

IFX assessments: RIA and RGA had lower limit of detection than ELISA (0.07 µg/mL and 0.13 versus 0.26). Maximal inaccuracies were 39%, 24%, and 23%. Imprecisions (coefficients of variation) were ≤20% within IFX concentrations between 1 and 9 µg/mL. All assays showed linear correlations (R = 0.97-0.99), but sample concentrations differed by up to 1.55 µg/mL for RIA and RGA, 1.41 µg/mL for ELISA and RIA, and 0.48 µg/mL for ELISA and RGA (P < 0.05). Anti-IFX Ab assessments: RGA gave highly reproducible results (coefficients of variation ≤ 7%) compared with all others (24%-26%). All assays had linear correlations (R = 0.71-0.93), except ELISA versus RGA and EIA. Assays disagreed on anti-IFX Ab titers with mean difference -420 (-1200 to 210) in RGA and EIA, and up to 4500 (-2700 to 11,800) in RIA and RGA. A contributing factor to these discrepancies was inability of ELISA to detect IgG4 anti-IFX Abs.

CONCLUSIONS::

Performances of assays for IFX and anti-IFX Abs are comparable. However, IFX concentrations and anti-IFX Ab titers show systematic differences, and in individual patients, only the same assay should be used. Problems may arise when different assays are used to manage therapies in the same patient.

INTRODUCTION:

: Tacrolimus has a large interindividual pharmacokinetic variability, and quantification of its effect is difficult. It is a substrate of ABCB1, an efflux pump expressed more on CD8 T cells than on CD4 T cells. The ABCB1 3435C>T single-nucleotide polymorphism (SNP) has been associated with interindividual differences in ABCB1 activity and may influence drug efficacy. Here the influence of this SNP on the biological effect of tacrolimus was studied.

METHODS::

Rhodamine (Rh123) efflux was used to study ABCB1 activity, with or without the addition of the ABCB1 inhibitor verapamil. Intracellular interleukin (IL) 2 production in T cells was used to measure the pharmacodynamic effect of tacrolimus after phorbol-12-myristate-13-acetate/ionomycin stimulation of whole blood. In addition, the ABCB1 genotype of 36 tacrolimus-treated renal transplant patients was related to ABCB1 activity and tacrolimus efficacy.

RESULTS::

The mean Rh123 efflux was higher in CD8 T cells compared with CD4 T cells: 40% versus 19% of cells, respectively (P < 0.001). Verapamil almost completely blocked Rh123 efflux (to 1.8% of CD4 T cells and 0.5% of CD8 T cells), whereas tacrolimus did not change Rh123 efflux. Tacrolimus 10 ng/mL reduced the production of IL-2 in CD4 and CD8 T cells by 28.9% and 45.4% (P < 0.05). Tacrolimus-mediated inhibition of IL-2 was enhanced by verapamil (P < 0.05). This effect on tacrolimus pharmacodynamics was associated with ABCB1 3435C>T SNP in renal transplant patients: verapamil reduced the percentage of IL-2-producing CD4 and CD8 T cells by 14% and 22% in patients with the CC genotype (P < 0.05) but not in patients with the TT genotype. Moreover, the ratio of tacrolimus C0 over the percent of IL-2-producing CD8 T cells in CC genotype patients was significantly higher compared with TT genotype patients (P < 0.05), showing a smaller pharmacodynamic effect in CC genotype patients.

CONCLUSION:

: The ABCB1 3435C>T SNP influences ABCB1 activity of T cells and the pharmacodynamic effect of tacrolimus in kidney transplant patients.

BACKGROUND::

Quetiapine has been recently approved as an add-on therapy in the treatment of major depressive disorders in the case of inadequate response to antidepressant monotherapy. Thereby the antidepressant potential is attributed to the N-demethylated metabolite norquetiapine (NQ). The aim of this cross-sectional analysis was to relate quetiapine (Q) doses to serum concentrations of Q and its active metabolite and clinical effects.

METHODS::

Data were obtained from patients who had been treated with different antidepressants and augmented under naturalistic conditions with Q for whom blood level measurements were requested.

RESULTS::

For this analysis, 105 depressed patients were included who had been augmented with Q. The mean daily doses of Q were 222 ± 125 mg. Doses correlated significantly (P < 0.001) with the highly variable serum concentrations of both Q and NQ. Median serum concentrations of Q and NQ were 46 ng/mL (25th to 75th percentile 20-91 ng/mL) and 59 ng/mL (25th to 75th percentile 26-133 ng/mL), respectively. Concentrations per dose ranged from 0.10 to 0.58 ng·ml·mg for Q and from 0.17 to 0.59 ng·ml·mg for NQ. Most patients (55%) received comedications in addition to the antidepressant drug and Q. According to the clinical global impressions scale, 60% of the patients were either much (36%) or very much improved (24%). Receiver-operating characteristic analysis revealed no significant differences of serum concentrations between responders and nonresponders for NQ (P = 0.835) but a trend for Q (P = 0.056).

CONCLUSIONS::

Due to marked variability of Q and NQ concentrations in the blood, therapeutic drug monitoring may be helpful to identify pharmacokinetic peculiarities. The lack of correlation between serum concentrations of NQ and clinical improvement casts doubts on the concept that NQ is the pharmacologically active principle for the augmentation therapy.

: We are describing a case of pediatric maltreatment. A 3-year-old boy was brought to the emergency room because of drowsiness that was caused by what his parents described as an 'accidental' intake of a powder contained in a plastic wrapper that was found in a park.: Urine immunochemical screening for drugs of abuse showed a positive result for opiate exposure. Despite the described 'accident,' the physician suspected abuse and ordered a hair analysis to verify possible intake of drugs of abuse. The child's hair was analyzed along its whole length for drugs of abuse using gas chromatography mass spectrometry in accordance with international guidelines.: Morphine and 6-acetylmorphine were identified, and the doctor informed the city's juvenile court. The boy's family was involved with social services for a period of observation to confirm suspected prolonged abuse. Hair analysis proves to be a useful tool for periodical examination of drug exposure to protect children from significant health and social risks.

: The risk of long-term chronic allograft nephropathy and graft loss after kidney transplantation is increased in patients with a high intrapatient variability in the clearance of tacrolimus. CYP3A5 genotype has a significant influence on the oral bioavailability of tacrolimus and is a potential influence on variability of exposure.: The study population consisted of 118 renal transplant recipients with stable renal function 12 months after transplant. The intrapatient variability of tacrolimus concentration was calculated. The patients were divided into low- and high-intraindividual variability groups using the median variability of tacrolimus clearance as the cutoff value.: No differences in baseline characteristics were observed between the expressers (n = 37) and nonexpressers (n = 81) except for ethnicity, which is in line with previous observations. Tacrolimus dose requirement was significantly higher in patients expressing CYP3A5, confirming earlier observations (P < 0.0001). However, intraindividual variability of tacrolimus clearance was not related to CYP3A5 genotype (P = 0.3331).: The intrapatient variability of tacrolimus clearance was not associated with CYP3A5 genotype in stable renal transplant recipients.

: For patients treated with citalopram, it was recently shown that serum concentrations above 50 ng/mL on day 7 of treatment are associated with an improved therapeutic outcome. The aim of this post hoc analysis was to calculate a potential cost-effectiveness of therapeutic drug monitoring (TDM) considering costs for hospitalization, medication, and drug analysis.: The study included patients with major depression. Weekly measurements of serum concentrations and assessments of psychopathology were conducted.: Fifty-five patients were included in this analysis. For patients with high citalopram serum concentrations (>50 ng/mL), the mean duration of hospitalization was 49 ± 20 days, and it was 72 ± 37 days (P = 0.03) in the group with low drug concentrations (<50 ng/mL). Considering daily costs for hospitalization of 250&OV0556;, the potential savings amounted to 5750&OV0556; per patient for the 23 days. Assuming that 11% of the variation of duration of hospitalization per patient were attributed to the serum concentration of the drug, the resulting savings were 633&OV0556; per patient. Considering the officially listed price of 21&OV0556; per TDM assay, total costs for weekly measurements over a period of 10 weeks of hospitalization were 210&OV0556;. In the groups with high and low serum concentrations, daily costs for citalopram medication were 3.00 ± 0.80&OV0556; and 2.42 ± 0.70&OV0556;, respectively (P = 0.002), and the mean number of comedications was nearly identical, that is, 1.87 ± 1.74 and 1.81 ± 1.86 drugs, respectively (P = 0.919).: The data taken together indicate that TDM-guided dosing of citalopram has the potential to be cost effective by reducing the length of hospitalization.

: Melatonin is synthesized in the pineal gland and is an important circadian phase marker, especially in the determination of sleep patterns. Both temporary and permanent abnormal sleep patterns occur in children; therefore, it is desirable to have methods for monitoring melatonin in biological fluids in the diagnosis and treatment of such disorders.: The objective of the study is to develop a liquid chromatography-tandem mass spectrometry method for the determination of melatonin in saliva and to apply it to monitoring salivary concentrations in children with sleep disorders.: A deuterated internal standard (d7-melatonin) was added to a diluted saliva sample (20 µL) in an autosampler vial insert, and 50 µL were injected. Plasticware was strictly avoided, and all glassware was scrupulously cleaned and then baked at 120°C for at least 48 hours to obtain satisfactory performance. Reverse-phase chromatography was performed on a C8 column using a linear gradient elution profile comprising mobile phases A (0.1% aqueous formic acid) and B (15% methanol in acetonitrile containing 0.1% formic acid), pumped at a total flow rate of 0.8 mL/min. The run time was 8 minutes. After atmospheric pressure chemical ionization, mass spectrometric detection was in positive ion mode. Mass detection was by selected reaction monitoring mode with the following mass transitions used for quantification: melatonin, m/z 233.0 → 173.8 and d7-melatonin, m/z 240.0 → 178.3.: Linearity (r > 0.999) was established from 3.9 to 1000 pg/mL. Imprecision (coefficient of variation percent) was less than 11%, and accuracy was 100-105% (7.0-900 pg/mL). The method was selective, and the mean (range) ratio of the slopes of calibrations in water to those in daytime saliva samples collected from 10 healthy adult subjects was 0.989 (0.982-0.997), indicating negligible matrix effects. The application of the assay was demonstrated in healthy adults and in children being clinically investigated for sleep disturbances.: A validated liquid chromatography-tandem mass spectrometry method suitable for monitoring salivary melatonin in children with circadian rhythm sleep disorders is reported. The method also has potential application to pediatric population pharmacokinetic studies using sparse sampling of saliva as the biological sample matrix.

: Drug of abuse consumption throughout pregnancy is a serious public health problem and an important economic cost to the health system. The aim of this work was to compare maternal interview and hair analysis to determine drug consumption throughout pregnancy and to study relations among maternal interview, hair results, and neonatal outcomes.: Two hundred nine mothers agreed to participate. After delivery, they were interviewed and a hair sample collected. Hair samples were segmented in trimesters and analyzed for 35 drugs [opioids, cocaine, amphetamines, Δ-tetrahydrocannabinol (THC), ketamine, methadone, antidepressants, benzodiazepines, and hypnotics; limits of quantification 5-100 pg/mg] and for ethyl glucuronide (limit of quantification 10 pg/mg) by liquid chromatography-tandem mass spectrometry. Statistical analysis was performed with χ test and t test.: In the interview, 4.3% mothers declared using illicit drugs during pregnancy (cocaine 1.4%, THC 2.9%, and opiates 1%), 3.3% medicines (methadone 1.9%, benzodiazepines 1.9%, and antidepressants 0.5%), 21.5% tobacco, and 13.7% alcohol. Hair analysis showed 15.4% prevalence in illicit drugs (cocaine 12.4%, THC 3.8%, opiates 1%, and ketamine 1%), 22.5% in medicines (methadone 3.3%, benzodiazepines 11%, antidepressants 9.1%, zopiclone 1%, and fentanyl 1.4%), and 3.9% in alcohol. Neonatal abstinence syndrome was developed in 8.1% newborns, all of them from mothers with high methadone-positive hair results (>926.2 pg/mg). Statistically significant lower newborn weight and length were found in neonates from declared smokers compared with nonsmokers (P < 0.05).: Maternal hair analysis showed to be more sensitive than maternal interview to detect drug use during pregnancy, except for alcohol. In this preliminary study, no statistically significant differences were found between exposed and nonexposed newborns to drugs, except for tobacco consumption.