With the introduction of the novel immunosuppressive agents sirolimus and everolimus, new, potentially more effective immunosuppressive regimens are undergoing clinical evaluation. Whereas the calcineurin inhibitors cyclosporin A (CsA) and tacrolimus suppress early activation of T lymphocytes through inhibition of cytokines such as interleukin 2, the primary target of sirolimus and everolimus is mammalian target of rapamycin (mTOR), a specific cell-cycle regulatory protein. The inhibition of mTOR leads to suppression of cytokine-driven T-lymphocyte proliferation (1). Because of their distinct modes of action, a calcineurin inhibitor and an mTOR inhibitor act synergistically to block acute allograft rejection (2)(3)(4). Current evidence (5) suggests that drug monitoring is necessary, not only for the calcineurin inhibitors, but also for the mTOR inhibitors. In contrast to the calcineurin inhibitors, however, there are currently no commercially available immunoassays for the latter two drugs. There is a need, therefore, for assays that can simultaneously quantify both the calcineurin and the mTOR inhibitors. Furthermore, a major shortcoming of the commercial immunoassays for CsA and tacrolimus is their cross-reactivity with unpredictable amounts of both active and inactive metabolites of the parent drugs in patient samples. In the case of CsA, no immunoassay has completely fulfilled the criteria recommended by a consensus panel (6).
New developments in immunosuppression protocols require that assays for calcineurin inhibitors provide broader dynamic ranges. On the one hand, C2 monitoring has been proposed for optimizing dosage of the CsA microemulsion (7), thereby necessitating quantification of CsA at concentrations up to 2500 μg/L: the lack of validated dilution protocols can be a problem for C2 monitoring (8). On the other hand, lower target ranges for CsA (5) and tacrolimus (9) are being investigated for long-term maintenance therapy to minimize the adverse side effects, in particular nephrotoxicity, of these drugs. Current immunoassays exhibit …
A juvenile, female renal transplant recipient suffered two acute rejection episodes: the first on posttransplant day 31 while taking cyclosporine, prednisone, and mycophenolate mofetil (MMF); and the second on posttransplant day 67, when she was taking tacrolimus, prednisone, and MMF. Dosage of MMF was initially started at 2 g/d (corresponding to 600 mg MMF/m2 twice daily.), but was reduced to 250 mg/d to 500 mg/d after severe diarrhea and a paralytic ileus on posttransplant day 16. During therapy with tacrolimus, prednisone, and MMF, predose plasma mycophenolic acid (MPA) concentrations varied from 1.1 mg/L to 8.2 mg/L (median 3.0 mg/L). On posttransplant day 91, a 12-hour pharmacokinetic profile was obtained. The concentrations of MPA and its metabolites were determined with a validated high-performance liquid chromatography (HPLC) procedure. After oral MMF (250 mg) administration, the MPA concentration showed an atypical decline from a predose concentration of 6.0 mg/L to a value of 3.8 mg/L at 75 minutes postdose, and 3.4 mg/L at 6 hours postdose, before returning to 6.0 mg/L after 12 hours. The 12-hour area under the concentration–time curve (AUC) values for MPA and its major metabolite the phenolic glucuronide MPAG were 55.1 mg·h/L and 800 mg·h/L, respectively. An unusually high concentration (12-h AUC, 165 mg·h/L) of the phenolic glucose conjugate of MPA was found. The apparent renal clearance of MPAG was only 2.2 mL/min. Her creatinine clearance was 30 mL/min. MPAG clearances have been reported to range from approximately. 5.5 mL/min to 35 mL/min at a creatinine clearance of approximately 30 mL/min in renal transplant recipients. The authors' findings suggest that conjugation and clearance of MPA through the kidney is strongly impaired in this patient. The relatively high predose MPA concentrations could result from an enhanced enterohepatic circulation of MPA and its metabolites.
The echinocandin caspofungin (CAS) is a novel antifungal drug with fungicidal in vitro activity against all Candida spp., which are the most frequent cause of fungal keratitis. Penetration of CAS through the cornea into the aqueous humor after topical administration was investigated.A CAS solution with a concentration of 7 mg/ml was applied onto each rabbit's cornea. Drug application after corneal epithelium abrasion was processed in different time intervals: single application with aqueous humor sampling after 1 and 2 h. In addition, after continuous application of CAS every 30 min, aqueous humor concentrations of CAS after 1, 2 and 5 h were analyzed by liquid-chromatography tandem mass spectrometry.Topical administration of CAS without corneal epithelium abrasion resulted in no detectable amounts of the drug in the aqueous humor. However, with corneal abrasion, after a single application, levels of 2.16 +/- 1.57 microg/ml (n = 6) were reached after 1 h and then decreased to 1.76 +/- 0.88 microg/ml (n = 2) after 2 h. After serial application every 30 min, the following intracameral levels of CAS were detected: after 1 h, 2.11 +/- 1.09 microg/ml (n = 6); after 2 h, 4.94 +/- 1.80 microg/ml (n = 5), and after 5 h, 3.45 +/- 2.11 microg/ml (n = 6).In the aqueous humor, therapeutic drug levels can be reached that cover the MICs of most fungi after epithelial abrasion. To achieve a sustained high level of CAS as an effective antifungal therapy for corneal keratitis, CAS should be administered topically every 30 min after removal of the corneal epithelium.
A large body of evidence currently documents the utility of the MEGX test for real-time assessment of liver function in transplantation, critical care medicine, and various experimental models (1). The test is based on the conversion of lidocaine to its deethylated metabolite monoethylglycinexylidide (MEGX), primarily through the hepatic cytochrome P450 system. In the standard MEGX test, an intravenous bolus of a small lidocaine dose (1 mg/kg) is administered over 2 min. Blood specimens are collected for serum MEGX determination both before and at 15 and/or 30 min after lidocaine administration. The most commonly used method to measure serum MEGX has been an automated fluorescence polarization immunoassay (Abbott Laboratories) with a detection limit of 3 μg/L. This test, however, is no longer commercially available. HPLC methods with ultraviolet detection (2)(3) and gas chromatographic (GC) procedures with ionization or nitrogen-phosphorous detection (4)(5) were originally reported, but all of these techniques had a limit of quantification >10 μg/L. Several studies have shown that transplant candidates with MEGX test results <10 μg/L have a particularly poor 1-year survival rate (6)(7)(8). An improved HPLC method with fluorescence detection (9) and a capillary GC method with nitrogen-phosphorus detection (10) were therefore developed that achieved an adequate analytical sensitivity with limits of detection of ∼1–2 μg/L. The disadvantage of the HPLC method with fluorescence detection is the necessity to derivatize MEGX.
Because of its flexibility and high specificity, liquid chromatography–tandem mass spectrometry (LC-MS-MS) is finding increasing application for the quantification of numerous analytes. We now describe a reliable, simple, sensitive, and rapid procedure for determining MEGX in serum by LC-MS-MS. This procedure also allows the simultaneous measurement of serum lidocaine concentrations in the same sample.
MEGX hydrochloride and lidocaine hydrochloride were kind gifts from Astra (Stockholm, Sweden). The internal …
Abstract Nimodipine prevents cerebral vasospasm and improves functional outcome after aneurysmal subarachnoid hemorrhage (aSAH). The beneficial effect is limited by low oral bioavailability of nimodipine, which resulted in an increasing use of nanocarriers with sustained intrathecal drug release in order to overcome this limitation. However, this approach facilitates only a continuous and not an on-demand nimodipine release during the peak time of vasospasm development. In this study, we aimed to assess the concept of controlled drug release from nimodipine-loaded copolymers by ultrasound application in the chicken chorioallantoic membrane (CAM) model. Nimodipine-loaded copolymers were produced with the direct dissolution method. Vasospasm of the CAM vessels was induced by means of ultrasound (Physiomed, continuous wave, 3 MHz, 1.0 W/cm 2 ). The ultrasound-mediated nimodipine release (Physiomed, continuous wave, 1 MHz, 1.7 W/cm 2 ) and its effect on the CAM vessels were evaluated. Measurements of vessel diameter before and after ultrasound-induced nimodipine release were performed using ImageJ. The CAM model could be successfully carried out in all 25 eggs. After vasospasm induction and before drug release, the mean vessel diameter was at 57% (range 44–61%) compared to the baseline diameter (set at 100%). After ultrasound-induced drug release, the mean vessel diameter of spastic vessels increased again to 89% (range 83–91%) of their baseline diameter, which was significant ( p = 0.0002). We were able to provide a proof of concept for in vivo vasospasm induction by ultrasound application in the CAM model and subsequent resolution by ultrasound-mediated nimodipine release from nanocarriers. This concept merits further evaluation in a rat SAH model. Graphical abstract
