Michal Figurski

Factors affecting Aβ plasma levels and their utility as biomarkers in ADNI

Previous studies of Aβ plasma as a biomarker for Alzheimer’s disease (AD) obtained conflicting results. We here included 715 subjects with baseline Aβ1-40 and Aβ1-42 plasma measurement (50% with 4 serial annual measurements): 205 cognitively normal controls (CN), 348 patients mild cognitive impairment (MCI) and 162 with AD. We assessed the factors that modified their concentrations and correlated these values with PIB PET, MRI and tau and Aβ1-42 measures in cerebrospinal fluid (CSF). Association between Aβ and diagnosis (baseline and prospective) was assessed. A number of health conditions were associated with altered concentrations of plasma Aβ. The effect of age differed according to AD stage. Plasma Aβ1-42 showed mild correlation with other biomarkers of Aβ pathology and were associated with infarctions in MRI. Longitudinal measurements of Aβ1-40 and Aβ1-42 plasma levels showed modest value as a prognostic factor for clinical progression. Our longitudinal study of complementary measures of Aβ pathology (PIB, CSF and plasma Aβ) and other biomarkers in a cohort with an extensive neuropsychological battery is significant because it shows that plasma Aβ measurements have limited value for disease classification and modest value as prognostic factors over the 3-year follow-up. However, with longer follow-up, within subject plasma Aβ measurements could be used as a simple and minimally invasive screen to identify those at increased risk for AD. Our study emphasizes the need for a better understanding of the biology and dynamics of plasma Aβ as well as the need for longer term studies to determine the clinical utility of measuring plasma Aβ.

Therapeutic Drug Monitoring of Mycophenolic Acid

Mycophenolic acid (MPA)-based therapies are widely used in combination with calcineurin inhibitors as maintenance immunosuppression for kidney transplant recipients (1). The two MPA therapies used in clinical transplantation are mycophenolate mofetil (MMF [brand name CellCept, Roche Pharmaceuticals, Nutley, NJ]) and mycophenolate sodium (MPS [brand name Myfortic, Norvartis Pharmaceuticals, Nutley, NJ]). MMF has been used for more than a decade and is a prodrug of MPA. The standard dosage of MMF in combination with cyclosporine (CsA) is 1 g given twice daily, although the dosage may be somewhat lower when co-administered with tacrolimus. MPS is an enteric coated form of MPA that was more recently introduced into the clinical arena. A dosage of 720 mg of MPS provides bioequivalence to a dosage of 1000 mg of MMF in kidney transplant patients (2). Immunosuppression afforded by MPA is achieved via reversible and uncompetitive inhibition of inosine monophosphate dehydrogenase (IMPDH), resulting in inhibition of guanine nucleotide biosynthesis (3,4). This consequently leads to suppression of both new DNA synthesis and other pathways that depend on a continuous supply of guanine nucleotide pool, such as T cell surface antigens and other glycosylated membrane proteins (4). Although there has been increased interest to incorporate MPA therapeutic drug monitoring into routine clinical practice (5–9), this has not yet become widespread in the United States for several possible reasons, including ( 1 ) lack of availability of US Food and Drug Administration–approved automated simple assays, ( 2 ) attainment of low rejection rates using empiric dosing of MMF in many maintenance immunosuppression regimens, ( 3 ) the complex pharmacokinetics (PK) of MPA, and ( 4 ) absence of overt organ toxicity. Here we discuss and review the pertinent information and study data regarding ( 1 ) our current understanding of MPA PK and major factors that can influence MPA clearance, ( 2 ) the performance …

An Age-Dependent Pharmacokinetic Study of Intravenous and Oral Mycophenolate Mofetil in Combination with Tacrolimus for GVHD Prophylaxis in Pediatric Allogeneic Stem Cell Transplantation Recipients

Acute graft-versus-host disease (aGVHD) still remains a major limiting factor following allogeneic stem cell transplantation (AlloSCT) in pediatric recipients. Mycophenolate mofetil (MMF), an uncompetitive selective inhibitor of inosine monophosphate dehydrogenase, is a new immunosuppressant agent without major mucosal, hepatic, or renal toxicity compared to other prophylactic aGVHD immunosuppressant drugs. Although there has been an extensive pharmacokinetic (PK) experience with MMF administration following solid organ transplantation in children, there is a paucity of PK data following its use in pediatric AlloSCT recipients. We investigated the safety and PK of MMF as GVHD prophylaxis following intravenous (i.v.) and oral (p.o.) administration (900 mg/m(2) every 6 hours) in conjunction with tacrolimus, after myeloablative (MA) and nonmyeloablative (NMA) conditioning and AlloSCT in 3 distinct age groups of pediatric AlloSCT recipients (0-6 years, 6-12 years, and 12-16 years). Mycophenolic acid (MPA) in plasma samples was measured either by high-performance liquid chromatography (HPLC) or liquid chromatography/mass spectrometry (LC/MS/MS) as we have previously described. Plasma samples were obtained at baseline and at 0.5, 1, 2, 3, 4, and 6 hours after i.v. dosing on days +1, +7, +14, and at 2 time points between day +45 and +100 after p.o. administration post AlloSCT. MPA PK analysis included AUC (0-6 hours), C(max), T(max), C(ss), V(ss), C trough (C(0)), CL, and T((1/2).) Thirty-eight patients, with a median age of 8 years (0.33-16 years), 20/18 M:F ratio, 21/17 malignant/nonmalignant disease, 17/21 MA: NMA conditioning, 16 of 22 related/unrelated allografts. Median time to myeloid and platelet engraftment was 18 and 31 days, respectively. Mean donor chimerism on day +60 and +100 was 83% and 90%, respectively. Probability of developing aGVHD grade II-IV and extensive chronic GVHD (cGVHD) was 54% and 34%, respectively. There was significant intra- and interpatient MMF PK variability. There was a significant increase in i.v. MPA area under the curve (AUC)(0-6 hour) and C(max) (P < .0003) and a significant decrease in CL(ss) (P < .002) and V(ss) (P < .001) on day +14 versus day +7. Children <12 years of age had a significant increase in i.v. MPA T(max) (P = .01), V(ss) (P = .028), and CL(ss) (P < .001) compared to the older age group. There was a trend in increased i.v. MPA CL(ss) following MA versus NMA conditioning (P < .054); i.v. and p.o. MMF administration (900 mg/m(2) every 6 hours) in combination with tacrolimus was well tolerated in pediatric AlloSCT recipients. There was a significant increase in MPA exposure on day +14 versus day +7, suggesting improved enterohepatic recirculation at day +14 post-AlloSCT. Children <12 years of age appear to have a significantly different MPA PK profile compared to older children and adolescents and may require more frequent dosing.

Qualification of a Surrogate Matrix-Based Absolute Quantification Method for Amyloid-β42 in Human Cerebrospinal Fluid Using 2D UPLC-Tandem Mass Spectrometry

The primary aims of this work were to: 1) establish a calibrator surrogate matrix for quantification of amyloid-β (Aβ)42 in human cerebrospinal fluid (CSF) and preparation of quality control samples for LC-MS-MS methodology, 2) validate analytical performance of the assay, and 3) evaluate its diagnostic utility and compare it with the AlzBio3 immunoassay. The analytical methodology was based on a 2D-UPLC-MS-MS platform. Sample pretreatment used 5 M guanidine hydrochloride and extraction on μElution SPE columns as previously described. A column cleaning procedure involved gradual removal of aqueous solvents by acetonitrile assured consistent long-term chromatography performance. Receiver-operator characteristic (ROC) curve and correlation analyses evaluated the diagnostic utility of UPLC-MS-MS compared to AlzBio3 immunoassay for detection of Alzheimers disease (AD). The surrogate matrix, artificial CSF containing 4 mg/mL of BSA, provides linear and reproducible calibration comparable to human pooled CSF as calibration matrix. Appropriate cleaning of the trapping and analytical columns provided every-day, trouble-free runs. Analyses of CSF Aβ42 showed that UPLC-MS-MS distinguished neuropathologically-diagnosed AD subjects from healthy controls with at least equivalent diagnostic utility to AlzBio3. Comparison of ROC curves for these two assays showed no statistically significant difference (p = 0.2229). Linear regression analysis of Aβ42 concentrations measured by this mass spectrometry-based method compared to the AlzBio3 immunoassay showed significantly higher but highly correlated results. In conclusion, the newly established surrogate matrix for 2D-UPLC-MS-MS measurement of Aβ42 provides selective, reproducible, and accurate results. The documented analytical performance and diagnostic performance for AD versus controls supports consideration as a candidate reference method.

Improved protocol for measurement of plasma β-amyloid in longitudinal evaluation of Alzheimer’s Disease Neuroimaging Initiative study patients

The interassay variability and inconsistency of plasma β‐amyloid (Aβ) measurements among centers are major factors precluding the interpretation of results and a substantial obstacle in the meta‐analysis across studies of this biomarker. The goal of this investigation was to address these problems by improving the performance of the bioanalytical method.

The Alzheimer’s Disease Neuroimaging Initiative 2 Biomarker Core: A review of progress and plans

We describe Alzheimers Disease Neuroimaging Initiative (ADNI) Biomarker Core progress including: the Biobank; cerebrospinal fluid (CSF) amyloid beta (Aβ1–42), t‐tau, and p‐tau181 analytical performance, definition of Alzheimers disease (AD) profile for plaque, and tangle burden detection and increased risk for progression to AD; AD disease heterogeneity; progress in standardization; and new studies using ADNI biofluids.

High-performance liquid chromatography-mass spectroscopy/mass spectroscopy method for simultaneous quantification of total or free fraction of mycophenolic acid and its glucuronide metabolites.

Measurement of unbound fractions of mycophenolic acid and its metabolites may prove useful in explaining the complicated pharmacokinetic and pharmacodynamic behavior of this drug as well as in therapeutic drug monitoring. We developed a reliable, accurate, and sensitive liquid chromatography-tandem mass spectrometric method for the simultaneous quantification of mycophenolic acid (MPA), MPA glucuronide (MPAG), and MPA acyl-glucuronide (AcMPAG), total or unbound, in plasma, urine, and tissue extract. This method uses a single internal standard, carboxy-butoxy ether of mycophenolic acid (MPAC), and involves a simple sample preparation step. Aliquots of plasma, urine, or dissolved tissue extract (100 μL) or plasma ultrafiltrate for free analytes (50 μL) are treated with acetonitrile/formic acid mixture (99.5/0.5 v/v) followed by centrifugation and dilution with water. The prepared samples are then injected onto an extraction column (Eclipse XDB-C18 12.5 × 4.1 mm; Agilent Technologies, Palo Alto, CA) and washed with mobile phase composed of acetonitrile/water/formic acid (10/89.5/0.5 v/v/v) at a flow rate of 2.8 mL/min. A switching valve is activated 1 minute after sample injection. The analytes are eluted onto the analytical column (Eclipse XDB-C18 150 × 4.1 mm; Agilent Technologies) with a gradient of 0.5% aqueous formic acid, methanol, acetonitrile, and water. We used a tandem mass spectrometer with electrospray ion source, in which the tandem mass spectroscopy transitions were (m/z): 338→207 for MPA, 438→303 for MPAC, and 514→303 for MPAG and AcMPAG. The dynamic ranges (lower limit of quantitation and upper limit of quantitation) were as follows: 0.05 to 30 mg/L for total MPA and 1 to 300 μg/L for free MPA; 0.5 to 300 mg/L of total MPAG and 0.2 to 60 mg/L for free MPAG; and 0.025 to 15 mg/L of total AcMPAG and 1 to 60 μg/L for free AcMPAG. The precision at lower limit of quantitation was in the range of 8.0% to 11.9% for all three total analytes and 13.8 to 18.7% for the free analytes. Accuracy at lower limit of quantitation was in the range of 100% to 105% for total and 97% to 99% for free analytes. Between-day precision of quality control samples was 4.0% to 6.3% for human plasma spiked with total analytes and 4.5% to 14.4% for spiked plasma ultrafiltrate for free analytes. Mean absolute recovery ranged from 98.5% to 101.7% for MPA (both total and free), from 78.1% to 103.4% for MPAG and from 91.5% to 110.4% for AcMPAG. No significant ion suppression was found under these conditions for any of the analytes. Carryover effect was found to be at a maximum level of 0.02%. This method was successfully applied to analyze over 11,000 samples for total analytes, and over 8000 samples for free analytes in plasma, and has been in operation for nearly 3 years without loss of performance.

Development of a predictive limited sampling strategy for estimation of mycophenolic acid area under the concentration time curve in patients receiving concomitant sirolimus or cyclosporine.

Limited sampling strategies for estimation of the area under the concentration time curve (AUC) for mycophenolic acid (MPA) co-administered with sirolimus (SRL) have not been previously evaluated. The authors developed and validated 68 regression models for estimation of MPA AUC for two groups of patients, one with concomitant SRL (n = 24) and the second with concomitant cyclosporine (n=14), using various combinations of time points between 0 and 4 hours after drug administration. To provide as robust a model as possible, a dataset-splitting method similar to a bootstrap was used. In this method, the dataset was randomly split in half 100 times. Each time, one half of the data was used to estimate the equation coefficients, and the other half was used to test and validate the models. Final models were obtained by calculating the median values of the coefficients. Substantial differences were found in the pharmacokinetics of MPA between these groups. The mean MPA AUC as well as the standard deviation was much greater in the SRL group, 56.4 ± 23.5 mg·h/L, compared with 30.4 ± 11.0 mg·h/L in the cyclosporine group (P < 0.001). Mean maximum concentration was also greater in the SRL group: 16.4 ± 7.7 mg/L versus 11.7 ± 7.1mg/L (P < 0.005). The second absorption peak in the pharmacokinetic profile, presumed to result from enterohepatic recycling of glucuronide MPA, was observed in 70% of the profiles in the SRL group and in 35% of profiles from the cyclosporine group. Substantial differences in the predictive performance of the regression models, based on the same time points, were observed between the two groups. The best model for the SRL group was based on 0 (trough) and 40 minutes and 4 hour time points with R2, root mean squared error, and predictive performance values of 0.82, 10.0, and 78%, respectively. In the cyclosporine group, the best model was 0 and 40 minutes and 2 hours, with R2, RMSE, and predictive performance values of 0.86, 4.1, and 83%, respectively. The model with 2 hours as the last time point is also recommended for the SRL group for practical reasons, with the above parameters of 0.77, 11.3, and 69%, respectively.

Pharmacokinetic monitoring of mycophenolic acid in heart transplant patients: Correlation the side-effects and rejections with pharmacokinetic parameters

BACKGROUND Mycophenolate mofetil is a commonly used immunosuppressant in heart transplantation but pharmacokinetic monitoring is not routinely done. We performed a prospective pilot multi-center trial in de-novo heart transplant recipients to evaluate the pharmacokinetics (PK) of mycophenolic acid (MPA) at multiple time points in the first year following transplant. MATERIAL/METHODS MPA trough and estimated area-under-the-curve (AUC) values were obtained at multiple visits from 21 enrolled patients. We attempted to correlate the side-effects and rejections with PK parameters. RESULTS MPA AUC and trough levels increased modestly over 12 months with substantial inter and intra patient variability. Cardiac rejection was associated with low MPA AUC values with a threshold of <36.2 mg×h/L during the first two post-transplant weeks. A threshold of 2-weeks average MPA trough level of 1.43 mg/L provided a sensitivity 82% and a specificity of 60%. CONCLUSIONS Adequate MPA levels are associated with decreased risk of allograft rejection. For patients with Cyclosporine co-immunosuppression, we propose an MPA trough of 1.4 mg/L and an MPA AUC of 36 mg × h/L as threshold values for dose adjustments. We recommend monitoring MPA levels at 1, 2 and 4 weeks, 6 months, 1 year and whenever an unexplained side-effect or allograft rejection occurs. Additional MPA AUC measurements are recommended when trough levels do not explain the clinical picture.

Prospective quality-control monitoring in the context of a clinical trial

enforce boundary smoothness. The technique’s validity was tested by comparing our results to the manual HarP reference segmentations on a sample of 100 subjects from the ADNeuroimaging Initiative (ADNI) database. We used a leave-one-out strategy to train and then test the segmentations. We used Dice similarity index for objects and correlation coefficients for volumes to verify compliance of automated to manual segmentations. Results: The results show that our method is bias-free with an average Dice similarity coefficient value of 0.803 and overall correlation coefficient of r1⁄40.95. Figure 1 compares the HC volumes computed from the segmentations made by anatomists (reference volumes) with the corresponding volumes computed from the automated segmentations (estimated segmentations). Two fits are shown, with and without intercept (fit parameters listed in Table 1). Both fits are virtually indistinguishable. Overlaid to a MRI image, the new contours often outperform those made by the anatomists in delineating the HC tissues in all three planes, due to the increased smoothness. Conclusions:Our automated segmentation algorithm was able to generate accurate HC volume measurements on a sample of the ADNI cohort that is heterogeneous in terms of ages, cognitive status, manufacturers, and atrophy levels. To our knowledge, this is the first study to assess the accuracy of an automated algorithm with the official release of HarP segmentation labels. Our results hold promise for the utilisation of automated segmentation in large clinical trials and in clinical practice where there is a growing need for biomarkers to support diagnosis and monitor progression of AD.