A validated HPLC-MS/MS method for simultaneously analyzing curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetra-hydrocurcumin and piperine in human plasma, urine or feces.

Background: The spice curcumin is supposed to have many different beneficial health effects. To understand the complete pharmacokinetics of curcumin we need an analytical method to determine curcumin and its metabolites in human plasma, urine or feces. We have developed an HPLC-MS/MS method for the simultaneous analysis of curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and piperine in human plasma, urine or feces. Methods: Sample pretreatment involved a simple liquid-liquid extraction with tert-butyl methyl ether. Conjugated curcumin and analogs can be measured after enzymatic hydrolysis. Reversed-phase chromatography with a linear gradient of 50-95% methanol in 0.1% formic acid was used. Total run time is 15 min. The method was validated with regards to stability, specificity, sensitivity, linearity, accuracy, repeatability and reproducibility. The applicability of the method was tested using actual patients samples. Results: The LLOQ in plasma, urine and feces for curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and piperine ranged from 1 to 5 nM. Whereas all compounds could be quantified on a linear range between 2 and 400 nM. Plasma and feces recovery of curcumin was 97.1 ± 3.7% and 99.4 ± 16.2%, whereas urine showed a recovery of 57.1 ± 9.3%. All compounds had acceptable in-between day or between day variability in the different matrixes. Conclusion: A HPLC-MS/MS method was developed and validated for the simultaneous quantification of curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and piperine in human plasma, urine or feces. This method will aid in critically verifying the pharmacokinetics of curcumin made by supplement manufacturers and help us to provide insight in the claimed bioavailability of curcumin supplements.

Tobramycin and vestibulotoxicity: retrospective analysis of four cases.

Over a course of 7 months, four patients developed vestibulotoxicity after treatment with intravenous tobramycin. Since vestibulotoxicity is a serious adverse effect which can be irreversible, an investigation was undertaken to determine if there was a cause for the toxicity and whether the quality of care had been inadequate. In this period, 26 patients with cystic fibrosis were treated with tobramycin according to valid guidelines, of which four experienced acute dizziness which disrupted their daily activities. Two patients experienced irreversible bilateral vestibular hypofunction and two unilateral loss of the right labyrinth, with decreasing dizziness over time. No apparent cause for the vestibulotoxicity was found in these four patients and the simultaneous occurrence was not due to a lack in quality of care. Symptoms of dizziness and balance disorders should be recognised by patients and caretakers at an early stage so additional diagnostics can be done to prevent further deterioration.

No drug-drug interaction between tezacaftor-ivacaftor and clofazimine: A case report.

In this case report the potential drug-drug interaction between cytochrome P450 (CYP) 3A4 substrates tezacaftor-ivacaftor and CYP3A4/5 inhibitor clofazimine is investigated in a patient with cystic fibrosis. Exposure to tezacaftor, ivacaftor and its metabolites was assessed by determination of the area under the plasma concentration versus time curve (AUC0-24 h for tezacaftor and AUC0-12 h for ivacaftor and its metabolite) before start of clofazimine and 8 and 115 days after start of clofazimine. The AUC-ratio at day 115 and pre-start was 1.09, 1.45 and 0.747 for ivacaftor, hydroxymethyl ivacaftor and tezacaftor, respectively. This case suggests that clofazimine exhibits no clinically relevant increase in exposure to tezacaftor and ivacaftor.

Conscious perception and the modulatory role of dopamine: no effect of the dopamine D2 agonist cabergoline on visual masking, the attentional blink, and probabilistic discrimination.

RATIONALE: Conscious perception is thought to depend on global amplification of sensory input. In recent years, striatal dopamine has been proposed to be involved in gating information and conscious access, due to its modulatory influence on thalamocortical connectivity. OBJECTIVES: Since much of the evidence that implicates striatal dopamine is correlational, we conducted a double-blind crossover pharmacological study in which we administered cabergoline-a dopamine D2 agonist-and placebo to 30 healthy participants. Under both conditions, we subjected participants to several well-established experimental conscious-perception paradigms, such as backward masking and the attentional blink task. RESULTS: We found no evidence in support of an effect of cabergoline on conscious perception: key behavioral and event-related potential (ERP) findings associated with each of these tasks were unaffected by cabergoline. CONCLUSIONS: Our results cast doubt on a causal role for dopamine in visual perception. It remains an open possibility that dopamine has causal effects in other tasks, perhaps where perceptual uncertainty is more prominent.

Differential metabolic effects of oral butyrate treatment in lean versus metabolic syndrome subjects.

BACKGROUND: Gut microbiota-derived short-chain fatty acids (SCFAs) have been associated with beneficial metabolic effects. However, the direct effect of oral butyrate on metabolic parameters in humans has never been studied. In this first in men pilot study, we thus treated both lean and metabolic syndrome male subjects with oral sodium butyrate and investigated the effect on metabolism. METHODS: Healthy lean males (n = 9) and metabolic syndrome males (n = 10) were treated with oral 4 g of sodium butyrate daily for 4 weeks. Before and after treatment, insulin sensitivity was determined by a two-step hyperinsulinemic euglycemic clamp using [6,6-2H2]-glucose. Brown adipose tissue (BAT) uptake of glucose was visualized using 18F-FDG PET-CT. Fecal SCFA and bile acid concentrations as well as microbiota composition were determined before and after treatment. RESULTS: Oral butyrate had no effect on plasma and fecal butyrate levels after treatment, but did alter other SCFAs in both plasma and feces. Moreover, only in healthy lean subjects a significant improvement was observed in both peripheral (median Rd: from 71 to 82 µmol/kg min, p < 0.05) and hepatic insulin sensitivity (EGP suppression from 75 to 82% p < 0.05). Although BAT activity was significantly higher at baseline in lean (SUVmax: 12.4 ± 1.8) compared with metabolic syndrome subjects (SUVmax: 0.3 ± 0.8, p < 0.01), no significant effect following butyrate treatment on BAT was observed in either group (SUVmax lean to 13.3 ± 2.4 versus metabolic syndrome subjects to 1.2 ± 4.1). CONCLUSIONS: Oral butyrate treatment beneficially affects glucose metabolism in lean but not metabolic syndrome subjects, presumably due to an altered SCFA handling in insulin-resistant subjects. Although preliminary, these first in men findings argue against oral butyrate supplementation as treatment for glucose regulation in human subjects with type 2 diabetes mellitus.

Can we spice up our Christmas dinner? : Busting the myth of the 'Chinese restaurant syndrome'.

BACKGROUND: Monosodium glutamate (MSG), also referred to as Vetsin or E621, is a flavour enhancer frequently used in Asian cuisine and abundantly present in the famous Chinese dish Peking duck. MSG is notorious for triggering the onset of the so-called 'Chinese restaurant syndrome' (CRS), a complex of unpleasant symptoms, which might include flushing, sweating and the onset of atrial fibrillation (AF). This study aims to determine the effects of MSG on the occurrence of AF. METHODS: We conducted a placebo self-controlled single-arm study in the Academic Medical Centre in Amsterdam. We included paroxysmal AF patients who reported a consistent onset of AF upon MSG intake. During three admissions, participants were subsequently administered: placebo, 1.5 g and 3 g MSG. If AF was recorded after the dose of 1.5 g MSG, patients were given another placebo instead of 3 g MSG. The primary outcome was the onset of AF registered by 24-hour Holter monitoring. The secondary outcomes were any other arrhythmia and the onset of CRS defined as two or more symptoms of CRS after MSG intake. RESULTS: Six men participated in the study. Both 1.5 g and 3 g MSG were unrelated to CRS, arrhythmias or AF occurrence. CONCLUSION: Peking duck can be put on the Christmas menu without risking guests to be admitted to the emergency department with new episodes of AF.

Metabolic acidosis caused by concomitant use of paracetamol (acetaminophen) and flucloxacillin? A case report and a retrospective study.

PURPOSE: A patient was identified with severe metabolic acidosis, a high anion gap and 5-oxoproline accumulation, probably caused by the simultaneous use of paracetamol (acetaminophen) and flucloxacillin. We wanted to investigate the necessity to control the interaction between both drugs with an automatic alert system. METHODS: To investigate the relevance of the interaction of paracetamol and flucloxacillin, a retrospective study was conducted. Data on paracetamol and flucloxacillin prescriptions and laboratory data (pH, Na+, HCO3-, Cl-, albumin and 5-oxoproline levels) were combined to assess the prevalence of acidosis, calculate the anion gap and analyse 5-oxoproline levels in clinically admitted patients using both drugs simultaneously. RESULTS: In the 2-year study period, approximately 53,000 admissions took place in our hospital. One thousand and fifty-seven patients used paracetamol and flucloxacillin simultaneously, of which 51 patients (4.8%) had a serum pH ≤ 7.35. One patient, the same patient as presented in the case report, had a high anion gap and a toxic level of 5-oxoproline. CONCLUSION: The prevalence of metabolic acidosis is very low and the only patient identified with the interaction was recognised during normal clinical care. We conclude that automatic alerts based on simultaneous use of paracetamol and flucloxacillin will generate too many signals. To recognise patients earlier and prevent severe outcomes, a warning system (clinical rule) based on paracetamol, flucloxacillin and pH measurement may be helpful. Early calculation of the anion gap can narrow the differential diagnosis of patients with metabolic acidosis and measurement of 5-oxoproline can explain acidosis due the interaction of paracetamol and flucloxacillin.

How to achieve safe, high-quality clinical studies with non-Medicinal Investigational Products? A practical guideline by using intra-bronchial carbon nanoparticles as case study.

BACKGROUND: Clinical studies investigating medicinal products need to comply with laws concerning good clinical practice (GCP) and good manufacturing practice (GMP) to guarantee the quality and safety of the product, to protect the health of the participating individual and to assure proper performance of the study. However, there are no specific regulations or guidelines for non-Medicinal Investigational Products (non-MIPs) such as allergens, enriched food supplements, and air pollution components. As a consequence, investigators will avoid clinical research and prefer preclinical models or in vitro testing for e.g. toxicology studies. THE AIM OF THIS ARTICLE IS TO: 1) briefly review the current guidelines and regulations for Investigational Medicinal Products; 2) present a standardised approach to ensure the quality and safety of non-MIPs in human in vivo research; and 3) discuss some lessons we have learned. METHODS AND RESULTS: We propose a practical line of approach to compose a clarifying product dossier (PD), comprising the description of the production process, the analysis of the raw and final product, toxicological studies, and a thorough risk-benefit-analysis. This is illustrated by an example from a human in vivo research model to study exposure to air pollutants, by challenging volunteers with a suspension of carbon nanoparticles (the component of ink cartridges for laser printers). CONCLUSION: With this novel risk-based approach, the members of competent authorities are provided with standardised information on the quality of the product in relation to the safety of the participants, and the scientific goal of the study.

[Severe phenytoin intoxication in patients with hypoalbuminaemia]. / Ernstige fenytoïne-intoxicatie bij patiënten met hypoalbuminemie.

Two patients, a 35-year-old woman and a 60-year-old man, developed severe neurological side effects during treatment with phenytoin: disorientation, myoclonia, hallucinations and drowsiness in the first patient and a comatose state in the second. The woman had spina bifida, a ventriculoperitoneal drain because of hydrocephalus, recurrent urinary-tract infection, and a history of status epilepticus. The man suffered from diabetic ketoacidosis complicated by epileptic convulsions. In both patients, the total phenytoin concentration in the blood was within the therapeutic range of 8-20 mg/l. However, both had low serum albumin concentrations, below 25 g/l. Low serum albumin levels are associated with increased concentrations of the free fraction of phenytoin. Toxic levels of free phenytoin were found: 4 and 8 mg/l, respectively, while the therapeutic range is 0.5-2 mg/l. The first patient recovered after treatment with phenytoin was stopped, after which she was placed on a lower dosage; the second patient died. When prescribing phenytoin to patients with hypoalbuminaemia, one should be aware of the risk of intoxication due to a high level of free phenytoin and consequently an increased risk of severe neurological side effects.

Development of luciferase tagged brain tumour models in mice for chemotherapy intervention studies.

The blood-brain barrier (BBB) is considered one of the major causes for the low efficacy of cytotoxic compounds against primary brain tumours. The aim of this study was to develop intracranial tumour models in mice featuring intact or locally disrupted BBB properties, which can be used in testing chemotherapy against brain tumours. These tumours were established by intracranial injection of suspensions of different tumour cell lines. All cell lines had been transfected with luciferase to allow non-invasive imaging of tumour development using a super-cooled CCD-camera. Following their implantation, tumours developed which displayed the infiltrative, invasive or expansive growth patterns that are also found in primary brain cancer or brain metastases. Contrast-enhanced magnetic resonance imaging showed that the Mel57, K1735Br2 and RG-2 lesions grow without disruption of the BBB, whereas the BBB was leaky in the U87MG and VEGF-A-transfected Mel57 lesions. This was confirmed by immunohistochemistry. Bioluminescence measurements allowed the visualisation of tumour burden already within 4 days after injection of the tumour cells. The applicability of our models for performing efficacy studies was demonstrated in an experiment using temozolomide as study drug. In conclusion, we have developed experimental brain tumour models with partly disrupted, or completely intact BBB properties. In vivo imaging by luciferase allows convenient follow-up of tumour growth and these models will be useful for chemotherapeutic intervention studies.

A simple and sensitive assay for the quantitative analysis of paclitaxel in human and mouse plasma and brain tumor tissue using coupled liquid chromatography and tandem mass spectrometry.

The development and validation of an assay for the determination of paclitaxel in human plasma, human brain tumor tissue, mouse plasma and mouse brain tumor tissue is described. Paclitaxel was extracted from the matrices using liquid-liquid extraction with tert-butyl methyl ether, followed by chromatographic analysis using an alkaline eluent. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicate that calibration standards in human plasma can be used to quantify paclitaxel in all tested matrices. In human samples, the validated range for paclitaxel was from 0.25-1000 ng ml(-1) using 200 microl plasma aliquots and from 5 to 5000 ng g(-1) using 50 microl tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma). In mice, the ranges were 1-1000 ng ml(-1) and 5-5000 ng g(-1) using 50 microl of mouse plasma and 50 microl of tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma), respectively. The method can be applied to studies generating only small sample volumes (e.g. mouse plasma and tumor tissue), but also to studies in human plasma requiring a lower limit of quantitation. The assay was applied successfully to several studies with both human and mouse samples.

Improved penetration of docetaxel into the brain by co-administration of inhibitors of P-glycoprotein.

P-glycoprotein (Pgp) in the blood-brain barrier limits the brain's uptake of many anticancer drugs. We have investigated whether the Pgp inhibitors cyclosporin A, valspodar (PSC833) and elacridar (GF120918) increase the accumulation of docetaxel in the brain. Pgp knockout mice served as a reference model for the complete absence or complete inhibition of Pgp. Plasma and tissues were analysed by high-performance liquid chromatography. Cyclosporin A, valspodar and elacridar significantly increased the brain concentrations of docetaxel in wild-type mice to 38%, 56% and 59%, respectively, of those achieved in Pgp knockout mice. Valspodar and cyclosporin A also increased the docetaxel concentration in plasma and other tissues by 2- and 3-fold, whereas elacridar did not change the clearance. All three inhibitors therefore inhibit Pgp in the blood-brain barrier. Elacridar increases the accumulation of docetaxel in the brain without significant effects on systemic exposure. Further clinical tests with this latter combination are warranted.

Bioanalysis of zosuquidar trihydrochloride (LY335979) in small volumes of human and murine plasma by ion-pairing reversed-phase high-performance liquid chromatography.

We have developed and validated a sensitive and selective method for the quantitative determination of the P-glycoprotein inhibitor zosuquidar (LY335979) in human and murine plasma using only 50 microl sample volumes. Sample pretreatment involved liquid-liquid extraction with tert-butyl methyl ether. Zosuquidar and the internal standard chlorpromazine were separated using a narrow bore column (2.1 mm x 150 mm) packed with 3.5 microm symmetry C(18) material. The mobile phase consisted of 38% (v/v) acetonitrile in 50mM ammonium acetate buffer pH 3.8 containing 0.005 M 1-octyl sulfonic acid and was delivered at 0.2 ml/min. Detection was performed with a fluorescence detector set at an excitation wavelength of 260 nm and an emission wavelength of 460 nm. The calibration curve was prepared in blank human plasma and was linear over the dynamic range (10-1000 ng/ml). The lower limit of quantitation was 20 ng/ml. The validation results showed that the assay was selective and reproducible. Within the range of the calibration curve the accuracy was close to 100% and within-day and between-day precision were within the generally accepted 15% range. This method was applied to study the pharmacokinetics of i.v. administered zosuquidar in mice. The sensitivity of the assay was sufficient to determine the drug concentration in plasma samples obtained up to 24 h after administration.

Bioanalysis and preliminary pharmacokinetics of the acridonecarboxamide derivative GF120918 in plasma of mice and humans by ion-pairing reversed-phase high-performance liquid chromatography with fluorescence detection.

We have developed and validated a sensitive and selective method for the determination of the P-glycoprotein modulator GF120918 in murine and human plasma. Chlorpromazine is used as internal standard. Sample pretreatment involves liquid-liquid extraction with tert-butyl methyl ether. Chromatographic separation is achieved by reversed-phase high-performance liquid chromatography using a Symmetry C18 column and detection was accomplished with a fluorescence detector set at excitation and emission wavelengths of 260 and 460 nm, respectively. The mobile phase consists of acetonitrile-50 mM ammonium acetate buffer, pH 4.2 (35:65, v/v). To achieve good separation from endogenous compounds and to improve the peak shape the counter-ion 1-octane sulfonic acid (final concentration 0.005 M) was added to the mobile phase. The lower limit of quantitation was 5.7 ng/ml using 200 microl of human plasma and 23 ng/ml using 50 microl of murine plasma. Within the dynamic range of the calibration curve (5.7-571 ng/ml) the accuracy was close to 100% and within-day and between-day precision were within the generally accepted 15% range. The stability of GF120918 was tested in plasma and blood from mice and humans incubated at 4 degrees C, room temperature, and 37 degrees C for up to 4 h. No losses were observed under these conditions. This method was applied to study the pharmacokinetics of orally administered GF120918 in humans and mice. The sensitivity of the assay was sufficient to determine the concentration in plasma samples obtained up to 24 h after drug administration.