Predicting Tacrolimus Concentrations in the Skin of Adult Kidney Transplant Recipients: A Feasibility Study.

Solid organ transplant recipients are at an increased risk of developing skin cancers due to chronic immunosuppression, particularly with calcineurin inhibitors. Tacrolimus is the most prescribed calcineurin inhibitor in this patient cohort, and understanding tacrolimus concentrations in the skin will facilitate the development of anti-cancer preventive and therapeutic strategies. Here, we show that in mice, tacrolimus blood levels peaked rapidly ∼1 h post last oral dose while skin levels rose more slowly and remained high for at least 6 h. Subsequently, tacrolimus skin and blood concentrations were assessed in 15 kidney transplant recipients. The mean age was 61 years, the average time post-transplant was 7 years (range 0-21 years) and 87% were male. The average skin sampling time post tacrolimus dosing was 6 h 32 min. Skin tacrolimus concentrations ranged from 7.1 ng/g to 71.2 ng/g and correlated with blood concentrations (r = 0.6). Mouse and human mean skin concentrations were in a similar range. Our data suggests that tacrolimus measurements in the blood may be used to approximate tacrolimus concentrations in the skin of kidney transplant recipients, and further exploited for the delivery of anti-cancer therapies designed to antagonize the immunosuppressive effects of tacrolimus in the skin.

Clinically-Relevant Rapamycin Treatment Regimens Enhance CD8+ Effector Memory T Cell Function In The Skin and Allow their Infiltration into Cutaneous Squamous Cell Carcinoma.

Patients receiving immunosuppressive drugs to prevent organ transplant rejection exhibit a greatly increased risk of developing cutaneous squamous cell carcinoma (SCC). However, not all immunosuppressive drugs confer the same risk. Randomised, controlled trials demonstrate that switching renal transplant recipients receiving calcineurin inhibitor-based therapies to mammalian target of rapamycin (mTOR) inhibitors results in a reduced incidence of de novo SSC formation, and can even result in the regression of pre-existing premalignant lesions. However, the contribution played by residual immune function in this setting is unclear. We examined the hypotheses that mTOR inhibitors promote the enhanced differentiation and function of CD8+ memory T cells in the skin. Here, we demonstrate that the long-term oral administration of rapamycin to achieve clinically-relevant whole blood drug target thresholds, creates a "low rapamycin dose" environment in the skin. While both rapamycin and the calcineurin inhibitor tacrolimus elongated the survival of OVA-expressing skin grafts, and inhibited short-term antigen-specific CD8+ T cell responses, rapamycin but not tacrolimus permitted the statistically significant infiltration of CD8+ effector memory T cells into UV-induced SCC lesions. Furthermore, rapamycin uniquely enhanced the number and function of CD8+ effector and central memory T cells in a model of long-term contact hypersensitivity provided that rapamycin was present during the antigen sensitization phase. Thus, our findings suggest that patients switched to mTOR inhibitor regimens likely experience enhanced CD8+ memory T cell function to new antigen-challenges in their skin, which could contribute to their lower risk of de novo SSC formation and regression of pre-existing premalignant lesions.

The serum hepcidin:ferritin ratio is a potential biomarker for cirrhosis.

BACKGROUND: Serum hepcidin concentration is potentially affected by inflammation and iron stores in chronic liver disease (CLD), but little is known about the relationship between hepcidin and the degree of hepatic fibrosis. We investigated the potential role of serum hepcidin as a biomarker of advanced liver disease. METHODS: Serum hepcidin was measured in 332 adults with CLD of varying aetiologies, 45 healthy and 50 non-liver disease patient controls. Liver biopsy data were available for 228 CLD subjects. RESULTS: Hepcidin was decreased in CLD patients compared with non-liver disease patient controls (P < 0.0001) but not healthy controls, and was lowest in those with cirrhosis (P < 0.0001). Serum hepcidin correlated with hepatic hepcidin mRNA expression in 91 biopsy samples available for genetic analysis (r = 0.68, P < 0.0001). Hepcidin also correlated positively with serum ferritin concentration, transferrin saturation, ALT, serum albumin and haemoglobin, but negatively with serum bilirubin. The hepcidin:ferritin ratio was significantly lower in CLD subjects compared with healthy and disease controls, and decreased with each increase in the stage of fibrosis and siderosis. The hepcidin:ferritin ratio was associated with progressive fibrosis on linear regression, and a value of less than 0.1 was independently associated with cirrhosis on logistic regression analyses (OR 5.54, P < 0.001). Receiver operating characteristic analysis showed the hepcidin:ferritin ratio was able to distinguish between F0 and F4 stages of fibrosis (area under receiver operating characteristic curve = 0.86). CONCLUSIONS: The hepcidin:ferritin ratio is reduced in relation to increasing fibrosis in CLD and the use of this ratio may have potential future diagnostic implications as a marker of cirrhosis.

Therapeutic drug monitoring of tacrolimus by liquid chromatography-tandem mass spectrometry: is it truly a routine test?

Therapeutic drug monitoring of tacrolimus by high-performance liquid chromatography-tandem mass spectrometry has become standard practice. We report on the long-term (4.5 years) use of one such method. Whole blood samples (25 µL) were treated with zinc sulphate (100 µL) and acetonitrile containing ascomycin (internal standard, 250 µL). A high-performance liquid chromatography-tandem mass spectrometer operating in positive ion mode with an electrospray interface was used. Chromatography was performed on a TDM C(18) cartridge column (10 mm × 2.1 mm, 10 µm, Waters) using a switch gradient. A total of 4029 batches were analyzed for tacrolimus; this comprised of 81950 analyses of which 61027 were patient samples. Calibration curves (1.0-50 µg/L) were run on 1765 occasions (mean r(2)=0.999; range r(2)=0.988-0.999). Inter-batch accuracy and imprecision of the method (2.5, 12.5 and 30.0 µg/L), when in routine use, was 97.6-98.5% and <8.0%, respectively (n=4031). Evaluation of the method against other methods in an external quality control scheme revealed good agreement by linear regression analysis (y=0.924x+0.196, r(2)=0.985). The percentage difference between our results and that of all methods revealed a mean bias of -6.3% and a range of -33.3% to 11.1%. During the evaluation period, four batch failures occurred (0.1% failure rate) and greater than 1000 samples per analytical column was achieved. In conclusion, the described method is ideally suited as a routine test for tacrolimus in the clinical setting.

The current role of liquid chromatography-tandem mass spectrometry in therapeutic drug monitoring of immunosuppressant and antiretroviral drugs.

Therapeutic drug monitoring of critical dose immunosuppressant drugs is established clinical practice and there are similar good reasons to monitor antiretrovirals. The aim of this article is to review the recent literature (last five years), with particular reference to the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS offers many potential advantages. The superior selectivity of LC-MS/MS over immunoassays for immunosuppressant drugs has been widely reported. Simultaneous measurement of a number of drugs can be performed. It is currently routine practice for the four major immunosuppressants (cyclosporin, tacrolimus, sirolimus and everolimus) to be simultaneously measured in whole blood. While up to 17 antiretroviral drugs have been simultaneously measured in plasma. The exquisite sensitivity of LC-MS/MS also provides the opportunity to measure these drugs in alternative matrices, such as dried blood spots, saliva, peripheral blood mononuclear cells and tissue. However, the clinical utility of measuring these classes of drugs in alternative matrices is still to be determined.

Evaluation of limited sampling strategies for mycophenolic acid after mycophenolate mofetil intake in adult kidney transplant recipients.

Multiple limited sampling strategies (LSSs) have been proposed for estimation of mycophenolic acid (MPA) area under the concentration-time curve from 0 to 12 hours postdose (AUC 0-12) after mycophenolate mofetil intake. The aim of this study was to provide summary information on all published LSSs for MPA and to evaluate their predictive performance in an independent population of kidney transplant recipients. Seventy-eight LSSs for MPA were identified. Sixty-nine full AUC profiles were collected from 45 subjects (25 cotreated with cyclosporine and 20 with tacrolimus). Predicted MPA AUC 0-12, calculated by applying the relevant concentration measurements within the LSS equations, was compared with full AUC calculated by using all concentration measurements in the linear trapezoidal rule. Four error indices (median prediction error, median percentage prediction error [MPPE], root median squared prediction error, and median absolute percentage prediction error [MAPE]) were used to evaluate bias and imprecision. Twelve of the 25 LSSs for cyclosporine-cotreated recipients and one of the 53 LSSs for tacrolimus-cotreated recipients displayed acceptable (less than 15%) bias and imprecision. In the cyclosporine group, two equations demonstrated the highest predictive power, one that used four time points in the first 6 hours postdose (r2 = 0.84, MPPE 1.6%, MAPE 7.8%) and one that used four time points in the first 4 hours postdose (r2 = 0.76, MPPE -0.8%, MAPE 10.2%). In the tacrolimus group, an equation that used two time points in the first 4 hours postdose was superior (r2 = 0.80, MPPE -3.0%, MAPE 13.6%). Application of the LSSs most appropriate for cyclosporine-cotreated patients to the tacrolimus-cotreated group resulted in clinically unacceptable bias and imprecision and vice versa. High variability in performance of LSSs highlights the importance of validating any LSS before applying it to an alternative population. Attention to comedication use is of particular relevance when choosing a LSS.

Measurement of urinary oxypurinol by high performance liquid chromatography-tandem mass spectrometry.

Oxypurinol is the active metabolite of allopurinol which is used to treat hyperuricaemia associated with gout. Both oxypurinol and allopurinol inhibit xanthine oxidase which forms uric acid from xanthine and hypoxanthine. Plasma oxypurinol concentrations vary substantially between individuals and the source of this variability remains unclear. The aim of this study was to develop an HPLC-tandem mass spectrometry method to measure oxypurinol in urine to facilitate the study of the renal elimination of oxypurinol in patients with gout. Urine samples (50 microL) were prepared by dilution with a solution of acetonitrile/methanol/water (95/2/3, v/v; 2 mL) that contained the internal standard (8-methylxanthine; 1.5 mg/L), followed by centrifugation. An aliquot (2 microL) was injected. Chromatography was performed on an Atlantis HILIC Silica column (3 microm, 100 mm x 2.1mm, Waters) at 30 degrees C, using a mobile phase comprised of acetonitrile/methanol/50 mM ammonium acetate in 0.2% formic acid (95/2/3, v/v). Using a flow rate of 0.35 mL/min, the analysis time was 6.0 min. Mass spectrometric detection was by selected reactant monitoring (oxypurinol: m/z 150.8-->108.0; internal standard: m/z 164.9-->121.8) in negative electrospray ionization mode. Calibration curves were prepared in drug-free urine across the range 10-200 mg/L and fitted using quadratic regression with a weighting factor of 1/x (r(2) > 0.997, n=7). Quality control samples (20, 80, 150 and 300 mg/L) were used to determine intra-day (n=5) and inter-day (n=7) accuracy and imprecision. The inter-day accuracy and imprecision was 96.1-104% and <11.2%, respectively. Urinary oxypurinol samples were stable when subjected to 3 freeze-thaw cycles and when stored at room temperature for up to 6h. Samples collected from 10 patients, not receiving allopurinol therapy, were screened and showed no significant interferences. The method was suitable for the quantification of oxypurinol in the urine of patients (n=34) participating in a clinical trial to optimize therapy of gout with allopurinol.

An investigation into the bias between liquid chromatography-tandem mass spectrometry and an enzyme multiplied immunoassay technique for the measurement of mycophenolic acid.

Mycophenolic acid is now the second most widely used immunosuppressant in solid organ transplantation. Overestimation of mycophenolic acid concentration is a recognized problem of immunoassay, and high-performance liquid chromatography with ultraviolet detection methods have long analysis times and a risk of analyte coelution which may compromise high sample throughput in a clinically meaningful time frame. A novel liquid chromatography-tandem mass spectrometry assay for mycophenolic acid was developed using very small (10 microL) sample volumes and evaluated in comparison with an established immunological assay. The enzyme mediated immunoassay showed a median positive bias compared with liquid chromatography-tandem mass spectrometry of 14.6%. Linear regression analysis showed a significant positive impact of bilirubin (r2 = 0.230) on bias with further increases of r2 to 0.261, 0.286, and 0.294 with the stepwise addition of creatinine, hematocrit, and gamma-glutamyl transpeptidase, respectively. The impact of comedication and transplant type depended on the patient population: analysis of all samples showed opposing effects to analysis of those samples lacking data with biochemical variables above. The liquid chromatography-tandem mass spectrometry method described in this report is capable of measuring mycophenolic acid concentrations in very small sample volumes and in a timely fashion without the significant overestimates characterizing enzyme mediated immunoassay measurements in patients with serologic features characterizing liver or renal graft rejection.

Multi-center evaluation of a commercial Kit for tacrolimus determination by LC/MS/MS.

OBJECTIVES: A multi-center evaluation (3 sites) of the LC/MS/MS MassTrak tacrolimus Immunosuppressants Kit (Kit) was undertaken. DESIGN AND METHODS: Ten aspects of the analytical performance of the Kit were investigated based on FDA and CLSI guidelines. RESULTS: The linear analytical range of the procedure was between 0.68 and 31.7ng/mL. Within-run and total imprecision were <6% and <8% (n=240), respectively. Recoveries of tacrolimus added to clinical samples that contained between 5 and 10ng/mL of tacrolimus before addition were 99, 102 and 105% at 5.0, 10 and 20ng/mL, respectively. Comparison of in-house and Kit procedures in samples from liver (n=50-58) or kidney (n=50 or 51) transplant recipients yielded method mean biases between -2.0 and +10.7% at 5 and 15ng/mL. CONCLUSIONS: This evaluation indicates that the Kit is suitable for the monitoring of tacrolimus in kidney and liver transplant recipients.

A HPLC-mass spectrometric method suitable for the therapeutic drug monitoring of everolimus.

We report here the validation of an HPLC-electrospray-tandem mass spectrometry method for the quantification of everolimus, an immunosuppressant drug. Whole blood samples (100 microl) were extracted by protein precipitation which involved sample pre-treatment with zinc sulphate followed by acetonitrile (containing internal standard, 40-O-(3'-hydroxy)propyl-rapamycin). HPLC was performed using a step-gradient at a flow rate of 0.6 ml/min on a Waters TDM C18 column (10 mm x 2.1mm I.D.) with a resultant chromatographic analysis time of 2 min. Mass spectrometric detection by selected reaction monitoring (everolimus m/z 975.5-->908.3; internal standard m/z 989.5-->922.3). The assay was linear from 0.5 to 40 microg/l (r2>0.994, n=11). The inter- and intra-day analytical recovery and imprecision for quality control samples (1.25, 12.5 and 30 microg/l) were 93.4-98.2% and <10.7%, respectively (n=10). At the lower limit of quantification (0.5 microg/l) the inter- and intra-day analytical recovery was 94.4-95.8% with imprecision of <14.1% (n=10). The absolute recovery of everolimus (6.5 microg/l) and internal standard (12.5 microg/l) was 96.5 and 88.3%, respectively (n=3). A comparison of our method against the mean of all HPLC methods for a series of samples from an external proficiency testing scheme revealed good correlation as shown by the regression analysis: y=0.973x+0.301 (r2=0.986, n=71). In conclusion, the method described is suited to the current requirements for therapeutic drug monitoring of everolimus.