Phase II Trial of Temsirolimus for Relapsed/Refractory Primary CNS Lymphoma.

PURPOSE: In this phase II study (NCT00942747), temsirolimus was tested in patients with relapsed or refractory primary CNS lymphoma (PCNSL). PATIENTS AND METHODS: Immunocompetent adults with histologically confirmed PCNSL after experiencing high-dose methotrexate-based chemotherapy failure who were not eligible for or had experienced high-dose chemotherapy with autologous stem-cell transplant failure were included. The first cohort (n = 6) received 25 mg temsirolimus intravenously once per week. All consecutive patients received 75 mg intravenously once per week. RESULTS: Thirty-seven eligible patients (median age, 70 years) were included whose median time since their last treatment was 3.9 months (range, 0.1 to 14.6 months). Complete response was seen in five patients (13.5%), complete response unconfirmed in three (8%), and partial response in 12 (32.4%) for an overall response rate of 54%. Median progression-free survival was 2.1 months (95% CI, 1.1 to 3.0 months). The most frequent Common Toxicity Criteria ≥ 3° adverse event was hyperglycemia in 11 (29.7%) patients, thrombocytopenia in eight (21.6%), infection in seven (19%), anemia in four (10.8%), and rash in three (8.1%). Fourteen blood/CSF pairs were collected in nine patients (10 pairs in five patients in the 25-mg cohort and four pairs in four patients in the 75-mg cohort). The mean maximum blood concentration was 292 ng/mL for temsirolimus and 37.2 ng/mL for its metabolite sirolimus in the 25-mg cohort and 484 ng/mL and 91.1 ng/mL, respectively, in the 75-mg cohort. Temsirolimus CSF concentration was 2 ng/mL in one patient in the 75-mg cohort; in all others, no drug was found in their CSF. CONCLUSION: Single-agent temsirolimus at a weekly dose of 75 mg was found to be active in relapsed/refractory patients with PCNSL; however, responses were usually short lived.

A simulated car-driving study on the effects of acute administration of levocetirizine, fexofenadine, and diphenhydramine in healthy Japanese volunteers.

OBJECTIVE: Antihistamines are often used for treating allergic rhinitis. However, many older antihistamines cause sedative side effects. The sedative effects of antihistamines on car-driving have been investigated. This has not been investigated for levocetirizine, a new-generation antihistamine, in Asian populations, and so we evaluated its sedative effects in healthy Japanese subjects. METHODS: In this double-blind, placebo-controlled, four-way crossover study, healthy volunteers received single doses of levocetirizine 5 mg, fexofenadine 60 mg, diphenhydramine 50 mg, and placebo at intervals of at least 6 days. Simple brake reaction time and choice brake reaction time task (CBRT), a lateral tracking (LT) task, and a multiple task, a mixture of CBRT and LT task, were used to compare driving performance between the four drugs. Subjective sedation was also assessed. RESULTS: The simple brake reaction time and CBRT, and the CBRT component of the multiple task, did not show any significant differences between the drugs. In contrast, the LT, both as a single parameter and as a component of the multiple task, showed significant differences between diphenhydramine and the newer-generation antihistamines in a manner that corresponds with subjective sedation. CONCLUSIONS: Levocetirizine and fexofenadine did not impair psychomotor performance in subjects performing simulated car-driving tasks, while diphenhydramine did impair psychomotor performance in the subjects. Copyright © 2016 John Wiley & Sons, Ltd.

Toxicological screening of human plasma by on-line SPE-HPLC-DAD: identification and quantification of acidic and neutral drugs.

A multi-analyte screening method for the quantification of 50 acidic/neutral drugs in human plasma based on on-line solid-phase extraction (SPE)-HPLC with photodiode array detection (DAD) was developed, validated and applied for clinical investigation. Acetone and methanol for protein precipitation, three different SPE materials (two electro-neutral, one strong anion-exchange, one weak cation-exchange) for on-line extraction, five HPLC-columns [one C18 (GeminiNX), two phenyl-hexyl (Gemini C6 -Phenyl, Kinetex Phenyl-Hexyl) and two pentafluorophenyl (LunaPFP(2), KinetexPFP)] for analytical separation were tested. For sample pre-treatment, acetone in the ratio 1:2 (plasma:acetone) showed a better baseline and fewer matrix peaks in the chromatogram than methanol. Only the strong anion-exchanger SPE cartridge (StrataX-A, pH 6) allowed the extraction of salicylic acid. Analytical separation was carried out on a Gemini C6 -Phenyl column (150 × 4.6 mm, 3 µm) using gradient elution with acetonitrile-water 90:10 (v/v) and phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients r ≥ 0.9950/0.9910 were obtained for 46/four analytes. Additionally, this method allows the quantification of 23 analytes for therapeutic drug monitoring. Limits of quantitation ranged from 0.1 (amobarbital) to 23 mg/L (salicylic acid). Inter-/intra-day precisions of quality control samples (low/high) were better than 13% and accuracy (bias) ranged from -14 to 10%. A computer-assisted database was created for automated detection of 223 analytes of toxicological interests. Four cases of multi-drug intoxications are presented.

Toxicological screening of human plasma by on-line SPE-HPLC-DAD: identification and quantification of basic drugs and metabolites.

An automated multi-analyte screening method for the identification and quantification of 92 drugs and metabolites based on on-line solid-phase extraction-high-performance liquid chromatography-diode array detection technique was developed and successfully validated. In addition, a database with 870 entries including UV-spectra, relative/retention times and response factors of toxicologically relevant compounds was created. Plasma samples (0.2 mL) were treated with methanol, diluted with buffer and on-line extracted (Strata X, 20 ×2 mm, 25 µm) at pH 9. Analytical separation was carried out on a Gemini NX column (150 ×4.6 mm, 3 µm) using gradient elution with acetonitrile-water (90:10,v/v) and 0.05 m potassium dihydrogen phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients ≥0.9950 were obtained for 78 analytes. As an additional benefit, the newly developed method allows the quantification of 42 analytes (e.g. antidepressants, neuroleptics and anticonvulsants) in a concentration range suitable for therapeutic drug monitoring. Limits of quantitation ranged from 0.02 mg/L (chlordiazepoxide) to 3.4 mg/L (mexiletine). Inter- and intra-day precisions of quality control samples (low/high) were better than 15% (zolpidem) and accuracy (bias) ranged from -11% (opipramol, venlafaxine) to 11% (venlafaxine, trazodone). Tests for carry-over and sample stability under different storage conditions were also performed and stability was adequate. Four cases of poisoning analysis are presented.

Brain histamine H1 receptor occupancy measured by PET after oral administration of levocetirizine, a non-sedating antihistamine.

OBJECTIVE: Histamine H1 receptor (H1R) antagonists often have sedative side effects, which are caused by the blockade of the neural transmission of the histaminergic neurons. We examined the brain H1R occupancy (H1RO) and the subjective sleepiness of levocetirizine, a new second-generation antihistamine, comparing fexofenadine, another non-sedating antihistamine, as a negative active control. METHODS: Eight healthy volunteers underwent positron emission tomography (PET) imaging with [(11)C]doxepin, a PET tracer that specifically binds to H1Rs, after a single oral administration of levocetirizine (5 mg), fexofenadine (60 mg) or placebo in a double-blind crossover study. Binding potential ratios and H1ROs in the cerebral cortices regions were calculated using placebo. Subjective sleepiness was assessed with the Line Analogue Rating Scale and the Stanford Sleepiness Scale. RESULTS: There was no significant difference between the mean brain H1RO after levocetirizine administration (8.1%; 95% CI: -9.8 to 26.0%) and fexofenadine administration (-8.0%; 95% CI: -26.7 to 10.6%). Similarly, subjective sleepiness was not significantly different between the two antihistamines and placebo. Neither subjective sleepiness nor plasma concentrations was significantly correlated with the brain H1RO of the two antihistamines. CONCLUSION: At therapeutic dose, levocetirizine does not bind significantly to the brain H1Rs and does not induce significant sedation.

The antioxidant idebenone fails to prevent or attenuate chronic experimental autoimmune encephalomyelitis in the mouse.

Oxidative stress and mitochondrial dysfunction appear to contribute to neurodegenerative processes during multiple sclerosis (MS). Thus, antioxidants may represent a therapeutic option for MS. The antioxidant idebenone was proven to be beneficial in Friedreich's ataxia and Leber's hereditary optic neuropathy, two disorders caused by mitochondrial alterations. Here we showed that idebenone protected neuronal HT22 cells from glutamate-induced death in vitro. However, in experimental autoimmune encephalomyelitis, idebenone failed to affect disease incidence or onset when applied preventively, or to reduce disease severity when applied therapeutically. Histopathological examination of CNS from idebenone treated mice showed no improvement in inflammation, demyelination, or axonal damage. Thus, we hypothesize that idebenone treatment will likely not benefit patients with MS.

Eight cases of fatal and non-fatal poisoning with Taxus baccata.

This paper describes two fatalities, three non-fatal intentional and three accidental oral ingestions of yew (Taxus baccata) leaves. In all cases the post-mortem external examinations showed no signs of violence. Internal examinations revealed small green, needle-like particles on the tongue, in the esophagus and in the stomach. Yew leaves were also identified in the stomach contents, whereas Taxus leaves were cut into small pieces and then ingested in one case. The analytical method used was based on a liquid-liquid-extraction under alkaline conditions followed by LC-MS/MS analysis (QTRAP 5500). Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100×3) mm. The analytical method allows the simultaneous identification and quantification of the commercially available yew alkaloids taxoids (m/z): paclitaxel (854.2→105.0/286.1), 10-deacetyltaxol (10-DAT: 812.2→105.0/286.1), baccatin III (BAC III: 604.0→105.0/327.0), 10-deacetylbaccatin III (10-DAB III: 562.1→105.0/327.0), cephalomannine [taxol B] (562.1→105.0/327.0) and of 3,5-dimethoxyphenol (3,5-DMP: 155.0→111.9/122.9) also encompassing the qualitative analysis of the alkaloidal diterpenoids (Q1→194.0/107.0); reference mass spectra obtained from a yew leaves extract: monoacetyltaxine (MAT: 568.4), taxine B (584.2), monohydroxydiacetyltaxine (MHDAT: 626.4), triacetyltaxine (TAT: 652.4), monohydroxytriacetyltaxine (MHTAT: 668.4). In both fatalities, paclitaxel, 10-DAT and cephalomannine were not identified in urine, cardiac and femoral blood but all taxoids and 3,5-DMP were present in stomach content and excreted into the bile. In urine, highest 3,5-DMP concentration was 7500 µg/L and 23,000 µg/L after enzymatic hydrolysis, respectively. In intentional and accidental poisonings, when electrocardiogram (ECG) examinations revealed ventricular tachycardia and/or prolonged QRS intervals, taxines were identified in plasma/serum, even after the ingestion of a few number of yew leaves, when 3,5-dimethoxyphenol was not even found. According to the data from one near-fatal intentional poisoning, elimination half-life of MAT, TAXIN B, MHDAT and MHTAT in serum was calculated with 11-13 h and taxines were detected up to t=+122 h post-ingestion of approximately two handfuls of yew leaves.

Liquid chromatography-tandem mass spectrometry for analysis of intestinal permeability of loperamide in physiological buffer.

Analysis of in vitro samples with high salt concentrations represents a major challenge for fast and specific quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To investigate the intestinal permeability of opioids in vitro employing the Ussing chamber technique, we developed and validated a fast, sensitive and selective method based on LC-MS/MS for the determination of loperamide in HEPES-buffered Ringer's solution. Chromatographic separation was achieved with an Atlantis dC18 column, 2.1 mm×20 mm, 3 µm particle size and a gradient consisting of methanol/0.1% formic acid and ammonium acetate. The flow rate was 0.7 ml/min, and the total run time was 3 min. For quantification, two mass transitions for loperamide and a deuterated internal standard (methadone-d(3)) were used. The lower limit of loperamide quantification was 0.2 ng/ml. This new LC-MS/MS method can be used for the detection of loperamide in any experimental setup using HEPES-buffered Ringer's solution as a matrix compound.

Acquired coagulopathy caused by intoxication with the superwarfarin-type anticoagulant rodenticide flocoumafen.

A 28 year-old heretofore healthy woman was transferred to our hospital with a two-month history of recurring episodes of bleeding. Administration of vitamin K and prothrombin complex concentrates in the transferring hospital had only temporarily corrected both the markedly elevated international normalized ratio (INR) and the prolonged activated partial thromboplastin time (aPTT). The patient's medical and family history revealed no reason for these abnormalities. Our laboratory analyses revealed a sustained deficiency of vitamin K-dependent clotting factors. Presence of an acquired inhibitor of clotting factors was excluded. Thus we suspected, intoxication with an anticoagulant rodenticide. Liquid chromatography-mass spectrometry (LC-MS/MS) revealed pharmacologically active concentrations of flocoumafen, a rodenticide belonging to the superwarfarin family, in the patient's serum. While the long elimination half-life of superwarfarins is well described in rodents, information on pharmacokinetics in humans is not yet available. Therefore, patient management was not limited to prolonged administration of vitamin K, but also included repeated measurements of flocoumafen serum levels. During follow-up visits, clotting tests remained normal and flocoumafen levels gradually decreased, reaching the limit of quantification after 48 days. Based on the repeated measurements of flocoumafen serum levels, a half-life of 6.7 days was estimated in our patient, which is in clear contrast to the 220 days reported in rodents. Thus, monitoring flocoumafen serum concentrations in affected patients may provide a rational basis for the duration of vitamin K substitution and adequate follow-up intervals.

Rapid quantification of tilidine, nortilidine, and bisnortilidine in urine by automated online SPE-LC-MS/MS.

The opioid tilidine is a prodrug which is hepatically metabolized to active nortilidine and bisnortilidine. Due to the increasing abuse of tilidine by drug users and the lack of a specific immunoassay, we developed an analytical method for the quantification of tilidine, nortilidine, and bisnortilidine in urine suitable for screening. In a following step, this method was used to establish data on excretion kinetics of the substances in order to evaluate the time window of detection after a single oral dose of tilidine/naloxone and also was applied to authentic urine samples from correctional facilities. Urine samples were mixed with internal standard solution and extracted on a weak cation exchanger at pH 6 using a Symbiosis Pico system. The chromatographic separation was achieved within a 3.5-min run time on a Phenylhexyl column (50 × 2.0 mm, 5 µm) via gradient elution (methanol and 0.2% formic acid) at a flow rate of 0.50 mL/min. The ESI-MS/MS was performed on a QTrap 3,200 in positive multiple reaction monitoring mode using two mass transitions per analyte. Validating the method resulted in a lower limit of quantification of 1.0 µg/L followed by a linear calibration range to 100 µg/L for each analyte (r(2) > 0.99). The analytical method allowed the detection of a single dose of a commercially available tilidine solution up to 7 days after administration. Using this highly sensitive method, 55 of 3,665 urine samples were tested positive.

Toxicological screening in urine: comparison of two automated HPLC screening systems, toxicological identification system (TOX.I.S.*) versus REMEDI-HS.

In this paper, the comparison of two automated high-performance liquid chromatography (HPLC) screening systems, a newly developed toxicological identification system (TOX.I.S.) versus the commercially available Remedi-HS (Bio-Rad), is presented. Urine samples from 405 cases screened positive for amphetamines, cocaine, and opiates by immunological assays and confirmed by GC-MS were analyzed with both systems. In more than 80% (TOX.I.S.) and 78% (Remedi-HS) of the cases (except for cocaine), the results obtained by both HPLC methods showed agreement with the earlier obtained results by immunoassay prescreening and gas chromatography-mass spectrometry (GC-MS). The evaluation showed that both automated HPLC methods led to comparable results and can be used alternatively. As the confirmation results for cocaine were rather poor (45% TOX.I.S., 54% Remedi-HS) in comparison to GC-MS, the TOX.I.S. was further optimized for the detection of the cocaine metabolite benzoylecgonine (BEC). The BEC method improved the detectability of BEC from 45% to 80%. Besides confirmation screening, the use of both systems in cases of acute intoxications was investigated. Information about basic compounds was obtained from urine screening by both systems, which therefore were useful as complementary techniques in the toxicological laboratory. The TOX.I.S. offers advantages such as common equipment, modern software, and higher versatility with the opportunity to establish additional methods in the system.

New screening method for basic compounds in urine by on-line extraction-high-performance liquid chromatography with photodiode-array detection.

A fully automated, qualitative screening HPLC method for the identification of basic compounds in urine has been developed. A 1-ml volume of urine was extracted by on-line extraction and separated on two coupled strong cation-exchange (SCX) columns (2 x LunaSCX, 150 mm x 4.6 mm, 5 microm) under isocratic conditions. The mobile phase consisted of a mixture of potassium dihydrogenphosphate buffer (pH 2.3) and acetonitrile. The use of photodiode-array detection (DAD, lambda = 190-800 nm) gave access to a library of approximately 2600 toxicologically relevant compounds. The validated method is reliable, simple and in addition successfully proven with the analysis of real biological specimen for the routine use.

Acute bromadiolone intoxication.

A 55-year-old man came to the hospital with a bleeding wound on his tongue. The coating of his tongue was green, and his sputum was red. Because an increased international normalized ratio-value was measured, a blood sample was sent to our laboratory with the suspicion of coumarin intoxication. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis confirmed the poisoning was by bromadiolone, with its maximum serum concentration at 440 microg/L. The analysis of further samples resulted in a calculated elimination half-life of 140 h. The analytical method described was developed for the determination and quantitation of bromadialone using LC-MS. This method is suitable for the simultaneous identification and quantitation of 10 indirect anticoagulants in human serum, which include five superwarfarins (brodifacoum, bromadiolone, difenacoum, difethialone, and flocoumafen) as rodenticides licenced in Germany and five other vitamin K antagonists (acenocoumarol, coumatetralyl, coumachlor, phenprocoumon, and warfarin). The method is based on an acidic (pH 4.2) liquid-liquid extraction followed by LC-ESI-MS analysis. Analytical separation was carried out using an Atlantis C18 column (2.1 x 20 mm, 3 microm). The mobile phase consisted of methanol/0.1% formic acid; the flow rate was 0.6 mL/min, and the time needed for analysis was 5 min. The lower limit of quantitation was 5 microg/L (signal-to-noise > 10).