Michael Deters

Simultaneous quantification of sirolimus, everolimus, tacrolimus and cyclosporine by liquid chromatography-mass spectrometry (LC-MS).

Abstract We developed a universal liquid chromatographymass spectrometry (LC-MS) assay with automated online extraction to quantify simultaneously the immunosuppressants sirolimus, everolimus, tacrolimus, and cyclosporine. Whole blood (300 μl) plus 6 μl 32-desmethoxyrapamycin (1 ng/μl) as internal standard was treated with 600 μl methanol/0.2 M ZnSO4 (80/20 v/v). After vortexing (30 s) and centrifugation (20000 g, 5 min) 50 μl of the supernatant were loaded on an extraction column, were washed by 0.35 ml/min water for 3 min and, after activation of a column-switching valve, were back-flushed by 0.25 ml/min methanol/ water (90/10 v/v) onto a C18 analytical column. After 22 min the extraction column was washed for 2 min with methanol and for 3 min with water before starting the next run. Column temperatures were kept at 33 °C. Sodium adduct ions [M+Na]+ ions were detected in the selected ion mode. For sirolimus, everolimus and tacrolimus the assay was linear from 0.3 to 200 μg/l and for cyclosporine from 5 to 1000 μg/l (all r2>0.999). Recovery of all immunosuppressants and the internal standard was >90% and in general, inter-day and intra-day precision was <10%. The simultaneous quantification of blood levels by LC-MS seems to be the method of choice in transplanted patients receiving multiple immunosuppressants.

Liquid chromatography/mass spectrometry for therapeutic drug monitoring of immunosuppressants

Abstract The benefit of therapeutic drug monitoring (TDM) of immunosuppressants like sirolimus, everolimus, tacrolimus, and cyclosporine for the clinical outcome of transplant recipients has been shown in numerous studies. For the monitoring of cyclosporine, tacrolimus and mycophenolic acid immunoassays are mostly used. However, these have the disadvantage that on one hand the antibodies used cross-react to a varying extent with metabolites and on the other hand different immunosuppressants cannot be measured simultaneously. No commercial immunoassays are available for the quantification of sirolimus and everolimus. In modern immunosuppressive regimens, two or even more different immunosuppressants are combined, which allows a dose reduction of each single component. Analytical laboratories will consequently be challenged with blood samples containing a variety of different immunosuppressants in very low concentrations. Therefore, the high sensitive and specific detection techniques like liquid chromatography/liquid chromatography–mass spectrometry (LC–LC/MS), liquid chromatography/mass spectrometry (LC/MS) and LC/MS–MS seem to be the methods of choice now and in near future. However, the user of these new applications have to be aware of some pitfalls, as well.

Everolimus/cyclosporine interactions on bile flow and biliary excretion of bile salts and cholesterol in rats.

As a possible explanation for everolimus/cyclosporine-induced hypercholesterolemia seen in transplant recipients, we investigated the interactions of the immunosuppressants everolimus and cyclosporine on bile flow and biliary excretion of bile salts and cholesterol in a subchronic bile fistula model in rats because biliary excretion is a main elimination route of cholesterol. After 2 weeks of daily treatment, everolimus (1 mg/kg i.p.) and cyclosporine (5 mg/kg i.p) decreased bile flow (−45 and −36%) and biliary excretion of bile salts (−34 and −54%) and cholesterol (−25 and −39%) and increased serum concentrations of cholesterol (+40 and +17%) and triglycerides (+220 and +110%). Bile salt serum concentration was elevated only by cyclosporine (+100%), and not by everolimus. Everolimus/cyclosporine slightly enforced the cyclosporine-induced hyperlipidemia but not reduction of bile parameters, while the cyclosporine-induced increase in bile salts in serum was totally prevented. From these results we conclude that bile salt synthesis could be impaired by everolimus, which could be one reason for everolimus-induced hypercholesterolemia.

Influence of cyclosporine on the serum concentration and biliary excretion of mycophenolic acid and 7-O-mycophenolic acid glucuronide.

The authors have investigated whether cyclosporine decreases the serum concentration of mycophenolic acid, the active principle of the immunosuppressant mycophenolate mofetil, and increases that of the inactive metabolite 7-O-mycophenolic acid glucuronide by reducing their enterohepatic recirculation. Rats were treated daily with methylcellulose (1.66 mL/kg PO) plus 0.9% NaCl (6 mL/kg IP), mycophenolate mofetil (20 mg/kg PO) plus 0.9% NaCl (6 mL/kg IP), methylcellulose (1.66 mL/kg PO) plus cyclosporine (5 mg/kg IP), and mycophenolate mofetil (20 mg/kg PO) plus cyclosporine (5 mg/kg IP). After 14 days a bile fistula was installed to measure the biliary excretion of the immunosuppressants and their metabolites. After 90 minutes blood was taken to determine their concentrations in blood or serum by HPLC. Cyclosporine significantly decreased the serum concentration of mycophenolic acid by 39% and increased, not significantly, that of 7-O-mycophenolic acid glucuronide by 53%. The biliary excretion of 7-O-mycophenolic acid glucuronide was significantly reduced by cyclosporine by 57%, whereas that of mycophenolic acid was not affected. Mycophenolate mofetil did not show a significant effect on either the blood concentration or the biliary excretion of cyclosporine and its metabolites AM1, AM9, AM1c, and AM4N. Cyclosporine significantly decreased the serum concentration of active mycophenolate acid and increased, not significantly, the serum concentration of inactive 7-O-mycophenolic acid glucuronide, presumably by reducing the biliary excretion of this inactive metabolite.

Sirolimus/cyclosporine/tacrolimus interactions on bile flow and biliary excretion of immunosuppressants in a subchronic bile fistula rat model.

The new immunosuppressive agent sirolimus generally is combined in transplant patients with cyclosporine and tacrolimus which both exhibit cholestatic effects. Nothing is known about possible cholestatic effects of these combinations which might be important for biliary excretion of endogenous compounds as well as of immunosuppressants. Rats were daily treated with sirolimus (1 mg kg−1 p.o.), cyclosporine (10 mg kg−1 i.p.), tacrolimus (1 mg kg−1 i.p.), or a combination of sirolimus with cyclosporine or tacrolimus. After 14 days a bile fistula was installed to investigate the effects of the immunosuppressants and their combinations on bile flow and on biliary excretion of bile salts, cholesterol, and immunosuppressants. Cyclosporine as well as tacrolimus reduced bile flow (−22%; −18%), biliary excretion of bile salts (−15%;−36%) and cholesterol (−15%; −47%). Sirolimus decreased bile flow by 10%, but had no effect on cholesterol or bile salt excretion. Combination of sirolimus/cyclosporine decreased bile flow and biliary bile salt excretion to the same extent as cyclosporine alone, but led to a 2 fold increase of biliary cholesterol excretion. Combination of sirolimus/tacrolimus reduced bile flow only by 7.5% and did not change biliary bile salt and cholesterol excretion. Sirolimus enhanced blood concentrations of cyclosporine (+40%) and tacrolimus (+57%). Sirolimus blood concentration was increased by cyclosporine (+400%), but was not affected by tacrolimus. We conclude that a combination of sirolimus/tacrolimus could be the better alternative to the cotreatment of sirolimus/cyclosporine in cholestatic patients and in those facing difficulties in reaching therapeutic ranges of sirolimus blood concentration.

Comparative study analyzing effects of sirolimus-cyclosporin and sirolimus-tacrolimus combinations on bile flow in the rat.

The new immunosuppressive agent sirolimus is combined in transplant patients with the cholestatic substances cyclosporin and tacrolimus. Nothing is known about possible cholestatic effects of these combinations. Therefore, we compared their effects on bile flow and on important bile parameters in an acute bile fistula model in rats. Cyclosporin reduced bile flow, biliary excretion of bile salts, cholesterol, and GSH to 20–40% of basal values. Sirolimus decreased bile flow to 50% and excretion of GSH to 30% of the initial conditions but had no effect on cholesterol and bile salt excretion. In contrast, tacrolimus increased bile flow to 120% and GSH excretion to 220% of the basal levels. Sirolimus/cyclosporin decreased bile flow and bile parameters to the same extent as cyclosporin alone. Sirolimus/tacrolimus reversed sirolimus-induced reduction of bile flow and GSH excretion and resulted in a normal bile salt and cholesterol excretion, thus it may be the better alternative in cholestatic patients.

Plant Exposures Reported to the Poisons Information Centre Erfurt from 2001–2010

At the moment, no recent study about plant exposures in Germany and in the federal states the Poisons Information Centre (PIC) Erfurt is serving is available. To get new information about important characteristics of plant exposures like the development of frequency, plants, age groups involved, circumstances of exposure, and symptom severity, we conducted a retrospective study including all human plant exposures reported to the PIC Erfurt over a 10-year period from the beginning of 2001 to the end of 2010. In total, 13 001 plant exposures were registered. While the absolute number of plant exposures discontinuously increased from 1110 in 2001 to 1467 in 2009, and decreased to 1157 in 2010, their relative frequency to all human exposures fell from 9.2 % in 2001 to 5.9 % in 2010. Age groups: children 87.5 % (toddler 60.0 %); adults 11.3 % (middle-aged adults 5.2 %). Gender: female 39.0 % and male 41.2 %. Circumstances: accidental 91.6 %, unknown 4.6 %, abuse 2.9 %, suicide 0.9 %. Severity of symptoms: none to slight 85.5 %, moderate 1.7 %, unknown 12.7 %, severe 0.1 % (in total 9, one 4-year-old girl, involved plant genera: Aconitum, Arum, Chelidonium, Datura, brugmansia, Dieffenbachia, Ricinus, 2 Taxus), fatal 0.03 % (in total 4, involved plant genera: 2 Aconitum, 2 Taxus). In comparison to all human exposures, the relative frequency of severe symptoms in accidental and intentional plant exposures by abuse was significantly lower but as high by suicide. The significant higher involvement of children resulted mainly in none or mild symptoms. Severe symptoms could mostly be observed in adults in intentional plant exposures or when poisonous plants were mistaken for eatable. Because some plant exposures resulted in severe symptoms and even death, their dangerousness should not be trivialised.

Angiotensin II antagonists - an assessment of their acute toxicity

Objective. In Germany, increasing prescription rates of angiotensin II antagonists resulted in rising enquiries to Poisons Information Centres (PICs) during the last decade. Therefore, we aimed to assess their acute toxicity for deriving triage recommendations. Methods: An observational case series with data collected retrospectively from eight PICs in Austria, Germany and Switzerland. Inclusion criteria were monoexposure, defined dose, and documented follow-up. Results. In total, 206 cases of exposures to angiotensin II antagonists were included (candesartan, 94; eprosartan, 3; irbesartan, 20; losartan, 26; olmesartan, 16; telmisartan, 18; and valsartan, 29). The median dose expressed as a multiple of their maximum daily dose for adults adjusted to body weight (MDDw) was 2.3 in children and 6.8 in adults. Patients involved were 150 children with a median age of 2 years and a median body weight of 13 kg and 56 adults with a median age of 47 years and a median body weight of 70 kg. Most children remained asymptomatic (82.7%), 16.7% developed minor symptoms. Only once, a low blood pressure of 60/40 mm Hg required intravenous fluids after ingestion of a 8.75-fold MDDw of candesartan by a 2.5-year-old toddler. Among adults, 53.6% remained asymptomatic while almost half of the patients suffered from minor (37.5%) or moderate (8.9%) symptoms. Conclusion. As no or only minor symptoms were observed after ingestion of less than a fivefold MDDw in both children and adults, only symptomatic patients and those who have ingested a fivefold MDDw or higher dose should be referred for medical assessment.