Serum sAPRIL: A potential tumor-associated biomarker to colorectal cancer

Objective

The purpose of our study was to investigate the serum levels of soluble a-proliferation-inducing ligand (sAPRIL) in patients with colorectal cancer (CRC), benign intestinal disease and healthy volunteers and explore the potential possibility of sAPRIL severing as a CRC biomarker.

Methods

Our investigation was conducted on 35 blood samples obtained from CRC patients, 32 blood samples from patients with benign intestinal diseases and 31 blood samples from healthy volunteers. The sAPRIL concentrations were examined by an enzyme-linked immunosorbent assay (ELISA) and data were analyzed with non-parametric Mann–Whitney U-test and X²-test. The correlation relationship between sAPRIL and CEA, as well as sAPRIL and CA19-9 was assessed by non-parametric Spearmen's correlation test, respectively.

Results

The median value of sAPRIL in the malignant group was 10.43 ng/mL, compared with those of the benign group (4.89 ng/mL) and control group (3.30 ng/mL), respectively, which had an obvious significance (P < 0.0003). Area under the receiver-operating characteristic (ROC) curve for sAPRIL was 0.854 (95% CI, 0.776–0.933). The optimal cut-off level of sAPRIL was 5.49 ng/mL. Serum sAPRIL had a positive correlation with CEA (r = 0.637, P = 0.000) and CA19-9 (r = 0.357, P = 0.008) in 35 patients with colorectal cancer. sAPRIL showed higher sensitivity (82.9%) than those of CEA (74.3%) and CA19-9 (65.7%) in CRC, respectively.

Conclusion

The results indicated that serum sAPRIL, as a potential biomarker, had a positive diagnostic value for colorectal cancer.     

Clinical evaluation of two novel biointact PTH(1–84) assays in hemodialysis patients

Objectives

In chronic kidney disease–mineral and bone disorder (CKD-MBD), most treatment decisions are guided by parathyroid hormone (PTH) levels. Here, we aimed at assessing the technical and clinical performance of two novel automated biointact PTH(1–84) assays, from Roche Diagnostics (Ro) and DiaSorin (DS), in hemodialysis patients.

Design and methods

We recorded demographics, dialysis treatment characteristics, pharmacotherapy for CKD-MBD and laboratory work-up. Statistical methods included Passing–Bablok, and multiple linear regression.

Results

121 patients, dialyzing on average for 3.5 years (range: 0.1–22.5), with serum phosphate 1.9 ± 0.6 mmol/L (mean ± SD), participated in the study. Median serum concentration for intact PTH was 223 ng/L (range: 5–2844), and for biointact PTH(1–84) was 136 ng/L (Ro; range: 1–1644), respectively 138 ng/L (DS; range: 4-1580). Both biointact assays were significantly correlated (r = 0.98; Ro = 0.87 × DS + 19.60). Bland–Altmann plots revealed an average bias ± 2 SD of 10 ± 27 ng/L below 200 ng/L, and − 32 ± 157 ng/L above 200 ng/L (Ro minus DS). The variably adjusted association between PTH and serum phosphate was very similar, regardless of the PTH assay, but this was not the case for PTH-derived measures (ratios biointact/intact; differences intact minus biointact). (Log)PTH concentrations as well as serum phosphate were significantly associated with serum creatinine, but only in patients with > 0 mL urine per day.

Conclusions

Results from Roche and DiaSorin biointact PTH(1–84) assays were well correlated, but showed increased deviations at higher concentrations. Biointact PTH(1–84) levels are roughly two third of intact PTH. The association between PTH and serum creatinine may depend on residual renal clearance of PTH and/or serum phosphate.     

Clinical and prognostic role of circulating MMP-2 and its inhibitor TIMP-2 in HCC patients prior to and after trans-hepatic arterial chemo-embolization

Background and aims

Trans-hepatic arterial chemo-embolization is the most commonly used treatment for unresectable hepatocellular carcinoma. The prognostic impact of tumor biomarkers has not therefore been evaluated in this treatment. Imbalance between matrix metalloproteinase-2 and tissue inhibitor metalloproteinase-2 is considered to play an important role in extracellular matrix remodeling and degradation. Higher serum levels of MMP-2 have been shown to predict a poor prognosis and shorter overall survival in HCC after TACE. The objective of this study was to evaluate the serum levels of MMP-2 and TIMP-2 in HCC patients before and after TACE to evaluate their clinical significance and usefulness as prognostic biomarkers.

Methods

MMP-2 and TIMP-2 levels were measured by ELISA in 75 HCC patients and 30 healthy controls. Sera MMP-2 and TIMP-2 were correlated with clinico-pathological features.

Results

The mean serum MMP-2 and TIMP-2 levels of HCC patients before TACE were 1700 ± 71 ng/mL and 89 ± 45 ng/mL respectively, significantly higher than that of the control group: 771 ± 60 ng/mL (p < 0.0001, t-test) and 25.7 ± 20 ng/mL respectively (p < 0.0001, t-test). A significant decrease of MMP-2 levels after 1 and 3 months compared to baseline time was observed (p < 0.0001), while with TIMP-2 a gradual increase in serum before and after TACE (p < 0.01) was detected. No significant correlation between serum MMP-2 levels and other clinico-pathological features was observed. Patients with serum MMP-2 > 1500 ng/mL (median value) had worse overall and recurrence-free survival compared with those with serum MMP-2 levels < 1500 ng/mL before treatment.

Conclusion

Higher serum MMP-2 levels and MMP-2/TIMP-2 ratio could predict poor prognosis after TACE, suggesting prognostic role of these biomarkers in HCC.     

25(OH)D3 in patients with ovarian cancer and its correlation with survival

Objectives

The aim of this study was to examine vitamin 25(OH)D3 concentration in ovarian cancer patients in relation to a pathological subtype of the tumor, FIGO stage, grading, menopause status and overall 5-year survival.

Design and methods

72 epithelial ovarian cancer patients aged 37–79, who undergone optimal cytoreductive surgery were enrolled to the study group. Serum 25(OH)D3 concentration was measured using an electrochemiluminescence immunoassay before surgery. Serum concentration of 25(OH)D3 was also measured in a group of 65 healthy non-obese women aged 35–65 years.

Results

In patients with ovarian cancer serum concentration of 25(OH)D3 was lower than in the reference group (12.5 ± 7.75 ng/mL vs 22.4 ± 6.5 ng/mL). No significant correlation was found between serum 25(OH)D3 concentration and histological subtype, grading, FIGO stage and menopausal status. The study group was divided into two subgroups and the survival curves were analyzed. Overall 5-year survival rate was significantly higher in the subgroup of patients with 25(OH)D3 concentration over 10 ng/mL compared to women with concentration below 10 ng/mL.

Conclusions

Low 25(OH) D3 concentration associated with lower overall survival rate might suggest for the important role of severe deficiency in more aggressive course of ovarian cancer. Testing for 25(OH)D in the standard procedure could help to find ovarian cancer patients with worse prognosis, who would benefit of special attention and supplementation.     

A patient with a high concentration of B-type natriuretic peptide (BNP) but normal N-terminal proBNP concentration: A case report

Objectives

A 61-year-old female presented with non-typical chest pain. High levels of plasma B-type natriuretic peptide (BNP; result 3188 ng/L, reference range < 100 ng/L, method Abbott Architect) were found, although she did not exhibit dyspnoea or other clinical symptoms of heart failure. Echocardiography did not provide an explanation for the elevated BNP concentrations. In follow-up, the chest pain complaints disappeared but BNP remained elevated at the same levels. The serum N-terminal proBNP (NT-proBNP) concentration appeared to be normal. This led us to doubt the accuracy of the BNP values.

Design and methods

Possible interference was investigated with BNP and NT-proBNP assays from different manufacturers, various (auto)antibody tests, sample dilutions, addition of mouse serum and polyethylene glycol (PEG) precipitation.

Results

BNP and NT-proBNP concentrations were normal when measured using all other (NT-pro)BNP immunoassays. Serial dilutions of sample and addition of mouse serum did not alter the results. Specific (auto)antibody tests were negative. However, PEG precipitation showed the presence of a high molecular weight immunoreactive protein.

Conclusions

We report a false positive BNP result possibly caused by a macro-BNP. This macro-BNP was only immunoreactive in the Abbott Architect BNP immunoassay. Clinicians should be aware of analytical interference when BNP results are constantly elevated in the absence of (non)cardiac causes.     

Reference intervals for serum creatinine levels in the healthy geriatric population

Background

There are few reliable reference values of enzymatically assayed serum creatinine (Scr) levels categorized within a small age interval in the healthy geriatric population. The aim of this study was to establish the reference intervals (RIs) for Scr in the elderly population.

Methods

Healthy, elderly Chinese Han ethnic individuals aged between 60 and 89 years old were recruited for this study. We stratified the reference individuals by gender and age (60–69, 70–79 and 80–89 years), and the Scr values were measured by an enzymatic method. The central 95 percentile RIs were determined using non-parametric statistical methods.

Results

The Scr values in the elderly population show a Gaussian distribution and age/sex related differences. The RIs for Scr in the reference population with respect to age (ranges of 60–69, 70–79 and 80–89 years) were 52.9–94.5, 57.3–106.2 and 59.0–110.8 μmol/L for males, respectively, and 44.3–75.4, 47.1–85.5 and 45.1–90.9 μmol/L for females, respectively.

Conclusions

We have established the RIs for Scr measured with an enzymatic method in the healthy Chinese Han ethnic elderly population, which can provide a reference for both clinical and laboratory studies.     

Single-Dose Pharmacokinetic Properties,Bioavailability, and Tolerability of Two Lamivudine 100-mg Tablet Formulations: A Randomized Crossover Study in Healthy Chinese Male Subjects

Background

Lamivudine is used in the treatment of HIV and chronic hepatitis B (HBV) infections. Since 1999, at least 2 million Chinese HBV patients have been treated with lamivudine, but there are limited studies on the pharmacokinetics and safety of the drug in Chinese populations.

Objective

This study was designed to assess the bioequivalence of a newly developed lamivudine tablet (test drug) and a branded lamivudine tablet (reference drug) in healthy Chinese male volunteers.

Methods

A single-center, single-dose, randomized, open-label, 2-period crossover study was conducted in 28 healthy Chinese male volunteers. Blood samples were collected up to 24 hours after the administration of oral lamivudine 100 mg in each period. Plasma lamivudine concentrations were analyzed by a validated LC–MS/MS method. Pharmacokinetic and bioavailability parameters were calculated. Adverse events (AEs) were recorded.

Results

There were no significant differences in mean (SD) pharmacokinetic parameters between the test and reference drugs, including C_max (1239 [328.9] ng/mL vs 1176 [341.5] ng/mL), AUC_0–t (4096 [599.1] ng · h/mL vs 4064 [678.2] ng · h/mL), and AUC_0–∞ (4200 [607.7] ng · h/mL vs 4162 [672.2] ng · h/mL). The geometric mean test/reference ratios (90% CI) calculated for the log-transformed parameters were C_max, 1.06 (96.21–116.90); AUC_0–t, 1.01 (96.53–105.39); and AUC_0–∞, 1.01 (96.81–105.16), all of which were within the acceptance limits for bioequivalence. No serious AEs were reported, and all mild AEs were recovered quickly without treatment.

Conclusion

These findings suggest that the test formulation of lamivudine 100 mg meets the FDA regulatory standards for bioequivalence with the reference formulation. Both formulations were well tolerated.     

Low-dose acetylsalicylic acid therapy monitored with ultra high performance liquid chromatography

Objectives

Assessment of compliance in patients on low-dose acetylsalicylic acid (ASA) therapy is limited to interview, pill counting, witnessed ASA intake, and measurement of thromboxane B₂ levels. We validated a new, sensitive assay for analyzing blood levels of ASA and the metabolite salicylic acid (SA).

Design and methods

Blood samples were withdrawn from 10 healthy volunteers and 50 patients with stable coronary artery disease, before and after intake of 75 mg ASA. Plasma was mixed with acetonitrile, and 2-methylbenzoic acid was added as internal standard. After filtration, ASA and SA were quantified with ultra high performance liquid chromatography (UHPLC). Separation was accomplished with an isocratic flow of acetonitrile in phosphate buffer pH 2.5, and a Zorbax RRHD C₁₈ analytical column. Detection was achieved with wavelength photodiode array detection set at 237 nm.

Results

The ASA- and SA-assay showed linearity (r² > 0.999) within the range of 0.2–200 μg/mL, and with detection limits of 170 ng/mL and 53 ng/mL. The intra- and inter-day imprecision for ASA and SA were 2.2–5.9%, 3.4–11.7% and 1.8–8.0%, 3.8–9.4%, respectively. Recovery was between 89 and 103% for both assays. More than 60 coadministered drugs were investigated for interference, and only one drug interfered with the ASA-assay and none with the SA-assay. ASA was measurable 1 h after intake of 75 mg ASA, and SA was measurable 1 and 6 h after ASA intake.

Conclusion

We developed a fast and reliable UHPLC assay with a high sensitivity and selectivity, suitable for monitoring of compliance in patients treated with low-dose ASA.     

A comparison of capture antibody fragments in cardiac troponin I immunoassay

Objectives

To compare cardiac troponin I (cTnI) values measured from 32 normal plasma specimens with a two-site cTnI research assay exploiting different molecular forms of a capture antibody.

Design and methods

The current research assay consists of two capture antibodies immobilized on streptavidin-well surface and one detection antibody attached to highly fluorescent europium(III)-chelate-doped nanoparticles. Four different molecular forms of one of the capture antibodies (intact monoclonal (Mab), F(ab′)₂ fragment, Fab fragment and chimeric Fab fragment (cFab)) were tested. The developed immunoassays were evaluated in terms of their analytical sensitivities and assay kinetics. Furthermore, cTnI concentrations were measured from 32 heparin plasma samples from apparently healthy donors (mean age 32; range 24–60 years).

Results

The differences in the measured cTnI concentrations (corrected for the buffer-based zero calibrator) between the Mab and the three fragmented forms were highly significant (P < 0.0001). Replacing the intact Mab with the antibody fragments also reduced the required antibody amount from 100 ng to 66 ng (F(ab′)₂) and 16.5 ng (Fab and cFab). Furthermore, the limit of detection was improved when Fab fragments were employed (Mab: 0.90 ng/L, Fab: 0.69 ng/L and cFab: 0.41 ng/L). The apparent normal range median (minimum/maximum) of the 32 healthy subjects was reduced from 7.28 ng/L (2.64/116 ng/L) with Mab to 1.80 ng/L (0.746/10.6 ng/L) for the cFab.

Conclusions

Eliminating the Fc-part from one of the two capture antibodies in an immunofluorometric cTnI assay substantially reduced the measured cTnI concentrations, simultaneously improving the assay sensitivity and reducing the reagent consumption.     

Simultaneous detection of 19 drugs of abuse on dried urine spot by liquid chromatography–tandem mass spectrometry

Background

The lack of specificity of immunoassays for drugs of abuse testing (DAT), and concerns over its cost in conjunction with reflex confirmatory tests prompted us to investigate the combinatorial use of dried urine spot (DUS) and LC–MS/MS as an alternative.

Methods

The method development and validation were performed in accordance with the guidelines published by FDA and CLSI.

Results

In this study we established and validated the precision, accuracy, and linearity of our DUS–LC–MS/MS method, and assessed the recovery, interference, and carryover as well. The linearity check for all 19 analytes demonstrated slopes between 0.94 and 1.04, and R² always greater than 0.99. Between-batch CV for QC at 4 difference levels ranged from 1.1% to 10%, where CV of LLOQ ranged from 1.2% to 12.8% and CV of ULOQ ranged from 0.8% to 5.1%. A concordance study with patient specimens between our method and GC–MS demonstrated 80.8% to 100% agreement. Stability of DUS specimens was assessed up to 30 days and the measured concentrations ranged from 94% to 114% of the 100 ng/mL urine calibrator used for this assessment.

Conclusions

We established and validated a DUS–LC–MS/MS method for DAT that conforms to the guidelines dictated by FDA, CLSI, and SAMHSA. While our method with high sensitivity and specificity provides an alternative diagnostic utility to EMIT immunoassays, it also offers superior solutions in specimen transportation, preservation, and storage. The benefits of our method are apparent in reducing turnaround time and test costs that result in better patient care.     

Age- and sex-specific reference values for fasting serum insulin levels and insulin resistance/sensitivity indices in healthy Iranian adults: Tehran Lipid and Glucose Study

Objectives

Increased insulin concentration is a surrogate for insulin resistance and early assessment of fasting insulin may help in identifying those who are potentially at high risk of type 2 diabetes, hypertension, and cardiovascular disease. The aim of this study was to determine age- and sex-related reference values for serum insulin and insulin resistance/sensitivity indices in Iranian subjects.

Design and methods

Serum insulin levels were measured by electrochemiluminescence immunoassay in 5786 participants of the Tehran Lipid and Glucose Study. After application of exclusion criteria, 309 non-obese healthy subjects (124 men and 185 women), aged 24–83 y, were included. The International Federation of Clinical Chemistry guidelines (non-parametric method) and the robust method were used for determining reference values.

Results

Overall 95% reference values for fasting insulin were 1.61–11.37, 2.34–11.98, and 2.11–12.49 μU/mL in men, women, and total population respectively. Mean fasting insulin concentration showed a decreasing trend with age in both genders (p for trend ≤ 0.001). Age, waist circumference, and systolic blood pressures were biological determinants of fasting insulin in both genders; in addition, insulin was modulated by triglycerides in men and fasting glucose in women. Reference intervals for HOMA1-IR, HOMA2-IR, and QUICKI were 0.63–2.68, 0.40–1.80, and 0.33–0.42, respectively.

Conclusion

This study presents the first set of reference values for fasting serum insulin to be 2–12 μU/mL for both genders in a healthy sample of Iranian adults along with the reference values for insulin resistance/sensitivity indices. These values could be used for identifying subjects with insulin resistance in epidemiological and clinical research.     

Reference values for fasting insulin in 75 year old females and males

Objectives

Reference intervals for insulin are often based on fairly small study groups with unknown body mass index (BMI). They are also much younger than most patients seeking care. These values are not optimal for elderly patients, as many biological markers change over time and adequate reference intervals are important for correct clinical decisions.

Design and methods

We studied fasting insulin (f-insulin) values in a cohort of 698 75-year old non-diabetic males and females. The 2.5th and 97.5th percentiles for all individuals, males and females were calculated according to the recommendations of the International Federation of Clinical Chemistry on the statistical treatment of reference intervals.

Results

There was a strong positive correlation between BMI and f-insulin, which led to the calculation of separate reference intervals for individuals with BMI ≤ 30.

Conclusions

The reference interval for f-insulin for all study subjects was 1.74–18.27 mIU/L and for individuals with BMI ≤ 30 (n = 574) the reference interval was 1.66–15.05 mIU/L.     

CUB and zona pellucida-like domain-containing protein 1 (CUZD1): A novel serological biomarker for ovarian cancer

Objectives

To measure the levels of serum CUB and zona pellucida-like domain-containing protein 1 (CUZD1) in patients with ovarian cancer (OvCa), benign gynecological conditions and healthy women and in a number of other cancer types (breast, colorectal, lung, prostate and testicular).

Design and methods

Serum CUZD1 levels were measured with a commercial enzyme-linked immunosorbent assay (ELISA). All specimens were analyzed in duplicate. Preliminary verification was performed in serum using 9 healthy women and 20 late stage (III–IV) OvCa patients. An independent cohort of serum samples was used to validate the verification results (18 late stage OvCa, 8 benign gynecological conditions and 8 healthy controls). The following specimens were used for the other cancer types of unknown stage—breast (n = 11), colorectal (n = 10), lung (n = 10), prostate (n = 15) and testicular (n = 10).

Results

Serum CUZD1 was significantly elevated in ovarian cancer patients (range 95–668 μg/L) as compared to healthy controls (range 0.7–2.5 μg/L). The independent cohort of OvCa samples confirmed the preliminary verification results. CUZD1 was also elevated in breast and lung cancer specimens and not in colorectal, prostate and testicular cancer specimens.

Conclusions

CUZD1 appears to be a highly promising novel serum biomarker for OvCa diagnosis. Its performance in the 2 independent cohorts examined, and in lung and breast cancer patients warrants further investigation.     

Serum calprotectin levels in elderly males and females without bacterial or viral infections

Objectives

Calprotectin is released from activated leukocytes and calprotectin can thus be used as a marker for leukocyte activation. Faeces calprotectin is not only used as a marker for inflammatory bowel disease but can also be used to detect leukocyte activation in other body fluids. The aim of the present study was to study serum calprotectin levels in non-infected elderly individuals to establish reference intervals for the marker.

Methods

Serum calprotectin was analyzed by immunoturbidimetry in 75 year old females and males without known infections. Individuals with CRP > 20 mg/L were excluded as this could indicate a subclinical infection. The calprotectin levels in the remaining 713 individuals were used to calculate reference values for this population. The Spearman rank correlations between calprotectin and 27 other laboratory biomarkers were also investigated.

Results

There was a strong positive Spearman rank correlation between calprotectin and CRP (p < 0.000001) and alkaline phosphatase (p < 0.000001). There were also significant negative correlations between calprotectin and ApoA1 and direct HDL-cholesterol.

Conclusions

The reference interval for serum-calprotectin for all study subjects was 0.3–2.6 mg/L. Leukocyte alkaline phosphatase contributes to serum alkaline phosphatase levels.     

Serum procalcitonin as a marker of post-cardiac arrest syndrome and long-term neurological recovery,but not of early-onset infections,in comatose post-anoxic patients treated with therapeutic hypothermia

Objective

To examine the relationship of early serum procalcitonin (PCT) levels with the severity of post-cardiac arrest syndrome (PCAS), long-term neurological recovery and the risk of early-onset infections in patients with coma after cardiac arrest (CA) treated with therapeutic hypothermia (TH).

Methods

A prospective cohort of adult comatose CA patients treated with TH (33 °C, for 24 h) admitted to the medical/surgical intensive care unit, Lausanne University Hospital, was studied. Serum PCT was measured early after CA, at two time-points (days 1 and 2). The SOFA score was used to quantify the severity of PCAS. Diagnosis of early-onset infections (within the first 7 days of ICU stay) was made after review of clinical, radiological and microbiological data. Neurological recovery at 3 months was assessed with Cerebral Performance Categories (CPC), and was dichotomized as favorable (CPC 1–2) vs. unfavorable (CPC 3–5).

Results

From December 2009 to April 2012, 100 patients (median age 64 [interquartile range 55–73] years, median time from collapse to ROSC 20 [11–30] min) were studied. Peak PCT correlated with SOFA score at day 1 (Spearman's R = 0.44, p < 0.0001) and was associated with neurological recovery at 3 months (peak PCT 1.08 [0.35–4.45] ng/ml in patients with CPC 1–2 vs. 3.07 [0.89–9.99] ng/ml in those with CPC 3–5, p = 0.01). Peak PCT did not differ significantly between patients with early-onset vs. no infections (2.14 [0.49–6.74] vs. 1.53 [0.46–5.38] ng/ml, p = 0.49).

Conclusions

Early elevations of serum PCT levels correlate with the severity of PCAS and are associated with worse neurological recovery after CA and TH. In contrast, elevated serum PCT did not correlate with early-onset infections in this setting.     

Reference intervals for whole blood viscosity using the analytical performance-evaluated scanning capillary tube viscometer

Objectives

This study was performed to establish the reference intervals for whole blood viscosity (WBV) using the analytical performance-evaluated scanning capillary tube viscometer (SCTV).

Design and methods

The analytical performance of the SCTV was evaluated using three different levels of QC materials and sixty human EDTA-blood samples. To establish the reference intervals for WBV, 297 healthy individuals (123 men and 174 women) were selected from 1083 subjects.

Results

Within-day precisions with QC materials and human whole blood and between-day precisions with QC materials were below 5.0%, 6.6% and 8.0% in CVs at all shear rates, respectively. Comparison tests between the SCTV and the Brookfield viscometer showed a significant correlation (R² = 0.972, p < 0.001). The reference intervals for WBV in healthy men were 3.66–5.41 cP at 300 s^− 1 and 23.15–36.45 cP at 1 s^− 1 while those in women were 3.27–4.32 cP at 300 s^− 1 and 18.20–27.36 cP at 1 s^− 1, respectively.

Conclusions

Using the analytical performance-evaluated SCTV, the reference intervals for WBV were established in healthy adults, which could be beneficial to the clinical utility of WBV in the aspect of appropriate modalities for the improvement of blood viscosity.     

Mycophenolic acid quantification in human peripheral blood mononuclear cells using liquid chromatography–tandem mass spectrometry

Background and objectives

Mycophenolic acid (MPA) is an immunosuppressant widely used to prevent organ rejection after organ transplantation. MPA therapeutic drug monitoring (TDM) is currently recommended by plasma C₀ – or even better – by AUC determinations, but little is known about the potential interest of measuring the intra-lymphocyte MPA concentrations, representing the target site of action. The aim of this study is to develop and validate a selective and sensitive analytical method for quantification of MPA levels in human peripheral blood mononuclear cells (PBMCs).

Methods

PBMCs were extracted from heparin blood samples by Leucosep®. Methanol and MPA-d3 were used as extraction solvent and internal standard, respectively. Chromatographic separation was obtained on a XBridge BEH C18 column (2.1 mm × 75 mm, 2.5 μm) maintained at 50 °C on a Waters Alliance 2795 coupled to a QuattroMicro tandem mass-spectrometer. The multiple reaction monitoring transitions used for quantification were m/z 320.97 → 207.0 for MPA, and 324.2 → 210.1 for MPA-d3, in positive ESI mode.

Results

The total HPLC run time was 6 min. The retention times for MPA-d3 and MPA were 2.55 for both compounds. The method was linear from 0.1 to 50 ng/mL MPA. The coefficient r² ranged from 0.996 to 0.998. Intra-assay and inter-assay imprecisions were < 10% in the whole range of concentrations, and < 20% at the LLOQ, and accuracy level was > 90%. The matrix and ion suppression effects were < 6%. The MPA limit of quantification was 0.1 ng/mL. No interference was identified in the assay. From the preliminary results, intra-cellular MPA concentrations in kidney transplant patients ranged from 0.69 to 3.39 ng/10⁷ cells.

Conclusion

We described a robust, rapid and simple method suitable for the determination of MPA concentrations in PBMCs. This method is currently used in pharmacokinetics–pharmacodynamics (PK–PD) clinical trials, in comparison to standard plasma TDM.     

Single-Dose,Randomized Crossover Comparisons of Different-Strength Imatinib Mesylate Formulations in Healthy Korean Male Subjects

Background

Imatinib mesylate is used to treat chronic myeloid leukemia and advanced gastrointestinal stromal tumors.

Objective

The purpose of this study was to compare the pharmacokinetics of 2 different strengths of the imatinib formulation containing 100 mg (reference) and 400 mg (test) to satisfy the regulatory requirement for marketing.

Methods

A single-center, randomized, single-dose, open-label, 2-period, 2-sequence, comparative crossover study with a 14-day washout period was conducted in 30 healthy male volunteers. Plasma samples for the drug analysis were collected up to 72 hours after drug treatment. Participants received either the reference (4 tablets of 100-mg imatinib) or the test (1 tablet of 400-mg imatinib) formulation during the first period and the alternative formulation during the second period. The safety profiles and tolerability of the 2 formulations were also assessed based on physical examinations, laboratory tests, a 12-lead ECG, and vital signs.

Results

Thirty participants were initially enrolled; their mean (SD) age, height, weight, and body mass index were 24.9 (2.0) years (range, 23–30 years), 174 (5) cm (range, 164–185 cm), 69.9 (2.0) kg (range, 54.1–87.4 kg), and 23.0 (2.0) kg/m² (range, 18.5–26.9 kg/m²); 28 healthy participants completed both treatment periods. Two subjects did not complete the study because they withdrew consent for personal reasons. The observed mean (SD) C_max, AUC_0–last, and AUC_0–∞ values for the reference formulation were 1792 (357) ng/mL, 28,485 (6274) ng · h/mL, and 29,079 (6371) ng · h/mL, respectively. Corresponding values for the test formulation were 1710 (312) ng/mL, 27,222 (4624) ng · h/mL , and 27,872 (4751) ng · h/mL. The geometric mean ratios (90% CIs) between the 2 formulations at the 400-mg dose of imatinib were 0.9579 (0.9054–1.0136) for C_max, 0.9652 (0.9174–1.0155) for AUC_0–last, and 0.9679 (0.9203–1.0179) for AUC_0–∞, respectively. During the study period, 6 adverse events (3 for the reference and 3 for the test formulation) were reported; all were transient, mild, and resolved completely during the treatment period. There were 4 cases of nausea and 1 case each of dizziness and oropharyngeal pain. Four adverse events were considered related to the study drugs.

Conclusions

The results showed that despite the different strengths of the 2 imatinib formations, the test and reference formulations both met the regulatory criteria for pharmacokinetic equivalence at a dose of imatinib 400 mg in these healthy Korean male subjects. Both imatinib formulations seemed to be generally well tolerated. ClinicalTrials.gov identifier: NCT01270984.     

The development and characterization of an ELISA specifically detecting the active form of cathepsin K

Objective

Cathepsin K plays essential roles in bone resorption and is intensely investigated as a therapeutic target for the treatment of osteoporosis. Hence an assessment of the active form of cathepsin K may provide important biological information in metabolic bone diseases, such as osteoporosis or ankylosing spondylitis.

Methods

Presently there are no robust assays for the assessment of active cathepsin K in serum, and therefore an ELISA specifically detecting the N-terminal of the active form of cathepsin K was developed.

Results

The assay was technically robust, with a lowest limit of detection (LOD) of 0.085 ng/mL. The average intra- and inter-assay CV% were 6.60% and 8.56% respectively. The dilution recovery and spike recovery tests in human serum were within 100 ± 20% within the range of the assay.A comparison of latent and active cathepsin K confirmed specificity towards the active form. Quantification of the levels of active cathepsin K in supernatants of purified human osteoclasts compared to corresponding macrophages showed a 30-fold induction (p < 0.001).In contrast, in serum samples from osteoporotic women on estrogen or bisphosphonate therapy and from ankylosing spondylitis patients no clinically relevant differences were observed.

Conclusion

In summary, we have developed a robust and sensitive assay specifically detecting the active form of cathepsin K; however, while it monitors osteoclasts with high specificity in vitro, it appears that circulating levels of active cathepsin K do not reflect bone changes under these circumstances.     

Association of post-cardioversion transcardiac concentration gradient of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) and inflammatory biomarkers to atrial fibrillation recurrence

Objectives

Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) has been shown to have both pro- and anti-apoptotic activities and is associated to better prognosis in heart failure. The aim of this study was to determine the transcardiac concentration gradient of sTRAIL and inflammatory biomarkers after AF cardioversion and assess their relation to AF recurrence.

Design and methods

We measured transcardiac gradients (coronary sinus concentration minus aortic root concentration) of sTRAIL, C-reactive protein (hsCRP) and interleukin-6 (IL-6) in patients with non-valvular AF after electrical cardioversion. Six-month AF recurrence was the study endpoint.

Results

There were no differences in sTRAIL and hsCRP concentrations in peripheral venous blood between patients with and without AF recurrence (p = 0.066 and 0.149, respectively), while IL-6 was higher in patients with recurrence (p = 0.032). Only sTRAIL showed a significant transcardiac gradient [3 pg/mL (IQR 1–4 pg/mL); p = 0.01]. sTRAIL gradient was 4 pg/mL (IQR 3–5 pg/mL) in patients without recurrence versus − 1 pg/mL (IQR − 2–1 pg/mL) in those with recurrence (p < 0.001). IL-6 (p = 0.281) and hsCRP (p = 0.979) aortic concentrations were not significantly different from coronary sinus concentrations. In multivariate analysis, sTRAIL transcardiac gradient (beta − 0.81, p = 0.004) remained a negative predictor of AF recurrence.

Conclusion

This study demonstrates the existence of a significant transcardiac sTRAIL concentration gradient in patients with non-valvular AF, inversely associated to AF recurrence. These results suggest production of sTRAIL by the heart and a protective role against substrate-altering processes in AF-prone atria. This could have implications for TRAIL-targeting therapies currently under development.