No abstract

Nonspecific quantitation of [C]sucrose in blood and brain has been routinely used as a quantitative measure of the in vivo blood-brain barrier (BBB) integrity. However, the reported apparent brain uptake clearance (K) of the marker varies widely (∼100-fold). We investigated the accuracy of the use of the marker in comparison with a stable isotope of sucrose ([C]sucrose) measured by a specific liquid chromatography-tandem mass spectrometry method. Rats received single doses of each marker, and the K values were determined. Surprisingly, the K value of [C]sucrose was 6- to 7-fold lower than that of [C]sucrose. Chromatographic fractionation after in vivo administration of [C]sucrose indicated that the majority of the brain content of radioactivity belonged to compounds other than the intact [C]sucrose. However, mechanistic studies failed to reveal any substantial metabolism of the marker. The octanol:water partition coefficient of [C]sucrose was >2-fold higher than that of [C]sucrose, indicating the presence of lipid-soluble impurities in the [C]sucrose solution. Our data indicate that [C]sucrose overestimates the true BBB permeability to sucrose. We suggest that specific quantitation of the stable isotope (C) of sucrose is a more accurate alternative to the current widespread use of the radioactive sucrose as a BBB marker.

Amoxicillin-clavulanate (A/C) is currently the most effective oral antimicrobial in treating children with acute otitis media (AOM), but the standard dosage of 90 mg amoxicillin/6.4 mg clavulanate/kg of body weight/day commonly causes diarrhea. We examined whether an A/C formulation containing lower concentrations of clavulanate would result in less diarrhea while maintaining plasma levels of amoxicillin and clavulanate adequate to eradicate middle-ear pathogens and to achieve clinical success. We conducted an open-label study in children with AOM who were 6 to 23 months of age. In phase 1, we treated 40 children with a reduced-clavulanate A/C formulation providing 90 mg amoxicillin/3.2 mg clavulanate/kg/day for 10 days. In phase 2, we treated 72 children with the same formulation at a dosage of 80 mg amoxicillin/2.85 mg clavulanate/kg/day for 10 days. We compared the rates of protocoldefined diarrhea (PDD), diaper dermatitis, and AOM clinical response in these children with rates we had reported in children who received the standard A/C regimen, and we obtained plasma levels of amoxicillin and clavulanate at various time points. Outcomes in phase 1 children and in children who had received the standard regimen did not differ significantly. Rates of PDD in children receiving phase 2 and standard regimens were 17% and 26%, respectively (P ϭ 0.10). The corresponding rates of diaper dermatitis were 21% and 33% (P ϭ 0.04) and of AOM treatment failure were 12% and 16% (P ϭ 0.44). Symptomatic responses did not differ significantly between regimens; both gave clavulanate levels sufficient to inhibit ␤-lactamase activity. In young children with AOM, clavulanate dosages lower than those currently used may be associated with fewer side effects without reducing clinical efficacy. (This study has been registered at ClinicalTrials.gov under registration no. NCT02630992.) KEYWORDS acute otitis media, amoxicillin-clavulanate, antimicrobial treatment N ext to the common cold, acute otitis media (AOM) is the most frequently diagnosed illness in children in the United States (1) and the most commonly cited indication for antimicrobial treatment (2). Findings of two recent placebo-controlled trials support routine antimicrobial treatment of AOM in very young children. In those trials, affected children under 3 years of age who received antimicrobial treatment for 7 or 10 days experienced more-favorable outcomes than those who received placebo (3, 4). In both studies, amoxicillin-clavulanate was used as the active treatment-albeit in different dosing regimens-because it had been previously shown to have the highest rates of eradication of AOM pathogens, with high resultant rates of clinical cure. The efficacy of A/C is attributable in part to inactivation, by the clavulanate

Accurate and reproducible measurement of blood-brain barrier (BBB) integrity is critical in the assessment of the pathophysiology of the central nervous system disorders and in monitoring therapeutic effects. The widely-used low molecular weight marker [(14)C]sucrose is non-specific in the absence of chromatographic separation. The purpose of this study was to develop and validate a sensitive and reproducible LC-MS/MS method for the analysis of stable isotope-modified [(13)C12]sucrose in brain, plasma, and blood to determine BBB permeability to sucrose. After addition of internal standard (IS, [(13)C6]sucrose), the marker and IS were recovered from diluted rat blood, plasma, and brain homogenate by protein precipitation using acetonitrile. The recovery of the marker and IS was almost quantitative (90-106%) for all three matrices. The recovered samples were directly injected into an isocratic UPLC system with a run time of 6 min. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear (r(2)≥0.99) in the concentration ranges tested for the diluted blood and plasma (10-1000 ng/mL) and brain homogenate (1-200 ng/mL). The lower limit of quantitation of the assay was 0.5 pg injected on column. The assay was validated (n=5) based on acceptable intra- and inter-run accuracy and precision values. The method was successfully used for the measurement of serial blood and plasma and terminal brain concentrations of [(13)C12]sucrose after a single intravenous dose (10 mg/kg) of the marker to rats. As expected, the apparent brain uptake clearance values of [(13)C12]sucrose were low in healthy rats. The method may be useful for determination of the BBB integrity in animal models.