A New Enzyme-Linked Immunosorbent Assay for a Total Anti-T Lymphocyte Globulin Determination: Development, Analytical Validation, and Clinical Applications.

BACKGROUND: Anti-T lymphocyte globulin (ATLG) modulates the alloreactivity of T lymphocytes, reducing the risk of immunological posttransplant complications, in particular rejection and graft-versus-host disease, after allogeneic hematopoietic stem cell transplantation (HSCT). We developed and validated a new enzyme-linked immunosorbent assay (ELISA) method to measure serum levels of total ATLG and evaluate the pharmacokinetics (PK) of the drug in children with ß-Thalassemia, receiving allogeneic HSCT. METHODS: Diluted serum samples were incubated with Goat-anti-Rabbit IgG antibody coated on a microtiter plate and then, with Goat-anti-Human IgG labeled with horseradish peroxidase. After incubation and washings, substrate solution was added and absorbance was read at 492 nm. ATLG concentrations in samples were determined by interpolation from a standard curve (range: 200-0.095 ng/mL), prepared by diluting a known amount of ATLG in phosphate-buffered saline (PBS). Low, medium, and high-quality control concentrations were 1.56, 6.25, and 25 ng/mL, respectively. This method was developed and validated within the acceptance criteria in compliance with the Guidelines for a biological method validation: the sensitivity of the method was 0.095 ng/mL. We analyzed serum samples from 14 children with ß-Thalassemia who received ATLG (Grafalon) at a dose of 10 mg/kg administered as intravenous (IV) infusion on days -5, -4, and -3 before HSCT (day 0). Blood sampling for PK evaluation was performed on days -5, -4, and -3 before and after drug infusion; and then from day -2 to +56. RESULTS: The median total ATLG levels pre-IVand post-IV were 0 and 118 mcg/mL on day -5; 85.9 and 199.2 mcg/mL on day -4; 153 and 270.9 mcg/mL on day -3, respectively. The median PK values of CL was 0.0029 (range: 0.0028-0.0057) L·kg·d, Vd was 0.088 (range: 0.025-0.448) L/kg and t1/2 was 20.2 (range: 5.8-50.2) days. CONCLUSIONS: These data suggest that given the marked interindividual variability of total ATLG disposition, the development of a validated ELISA method and the possibility to measure PK parameters in paediatric populations are essential steps to optimize drug therapeutic regimens.

Bioequivalence of a New Oral Levosulpiride Formulation Compared With a Standard One in Healthy Volunteers.

BACKGROUND: A monocentric, single-dose, open-label, 2-way, crossover randomized study was conducted by the San Matteo Phase I Clinical Trial Unit and Experimental Therapy (Pavia, Italy) to assess the bioequivalence and the systemic tolerability of a new oral formulation of levosulpiride (tablet 25 mg: test) versus a commercially available formulation on the Italian market (tablet 25 mg: reference). METHODS: Thirty-five healthy adult volunteers, men (n = 19) and women (n = 16), aged between 18 and 55 years were screened and 32 of them were enrolled in the study. After having signed the written informed consent, each subject received a single oral dose of Test or Reference product with 250 mL of natural mineral water, in fasting conditions, interspersed with a 6-day washout period Blood samples were collected up to 36 hours after drug administration: the drug plasma levels were determined by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The pharmacokinetic parameters included peak plasma concentration (Cmax), time corresponding to Cmax (tmax), area under the plasma concentration-time curve from zero to infinity (AUC0-∞) or to the last sampling time assessment (AUC0-36), the elimination rate constant (ke), and the terminal half-life (t1/2). Safety was measured by pre- and post-treatment specific biochemical investigations, physical examination, electrocardiogram, occurrence of adverse events, and any information on patients' withdrawal. RESULTS: The geometric mean ratio Test/Reference (90% confidence interval) for levosulpiride was 103.0% (95.8-110.8) for AUC0-36, 103.6% (95.9-111.9) for AUC0-∞, and 104.3% (94.9-114.6) for Cmax. ke and t1/2 were 0.07 (SD: 0.02) and 9 hours (8-12) for both the formulations. Clearance (L/h) was 29.6 (±13.5) and 30.7 (±14.2) for the test and the reference product, respectively. CONCLUSIONS: Because the acceptance criteria required by the drug regulatory agency (European Medicines Agency, EMA) for bioequivalence prescribe limits of 80%-120% for untransformed data and 80%-125% for "ln" transformed data, we can confirm that the 2 formulations are bioequivalent, in terms of the rate and extent of absorption.

Rituximab in children with resistant idiopathic nephrotic syndrome.

Idiopathic nephrotic syndrome resistant to standard treatments remains a therapeutic dilemma in pediatric nephrology. To test whether the anti-CD20 monoclonal antibody rituximab may benefit these patients, we conducted an open-label, randomized, controlled trial in 31 children with idiopathic nephrotic syndrome unresponsive to the combination of calcineurin inhibitors and prednisone. All children continued prednisone and calcineurin inhibitors at the doses prescribed before enrollment, and one treatment group received two doses of rituximab (375 mg/m(2) intravenously) as add-on therapy. The mean age was 8 years (range, 2-16 years). Rituximab did not reduce proteinuria at 3 months (change, -12% [95% confidence interval, -73% to 110%]; P=0.77 in analysis of covariance model adjusted for baseline proteinuria). Additional adjustment for previous remission and interaction terms (treatment by baseline proteinuria and treatment by previous remission) did not change the results. In conclusion, these data do not support the addition of rituximab to prednisone and calcineurin inhibitors in children with resistant idiopathic nephrotic syndrome.

Relationship between pharmacokinetic profile of subcutaneously administered alemtuzumab and clinical response in patients with chronic lymphocytic leukemia.

Alemtuzumab serum levels and clinical response after subcutaneous administration (10 mg 3 times/week for six weeks) have been explored in 29 chronic lymphocytic leukemia patients receiving the monoclonal antibody as consolidation. Serum concentrations after each administration gradually increased during the first week and more markedly during weeks 2 and 3, approaching the steady-state at week 6. Absorption continued slowly through the tissues for about 2-3 weeks after the last administration, starting to decrease thereafter. Difference between Responders and Non-responders was statistically significant: maximal concentration (Cmax) was 1.69 µg/mL vs. 0.44 µg/mL; concentration before subcutaneous administration (Cpre-dose) on day 15 was 0.7 vs. 0.21 µg/mL, area under curve (AUC0-12h) was 11.09 vs. 2.26 µgxh/mL for Responders and Non-responders, respectively. Higher systemic exposure to alemtuzumab correlated with a better clinical response and minimal residual disease. Results suggest that an adjusted schedule according to serum level could improve clinical outcome of patients receiving subcutaneous alemtuzumab.

Lower dose rituximab is active in adults patients with idiopathic thrombocytopenic purpura.

Rituximab 375 mg/m(2) weekly for four weeks has significant activity in patients with immune thrombocytopenia. We evaluated the activity of lower dose rituximab (100 mg iv weekly for 4 weeks) in 28 adults with idiopathic thrombocytopenic purpura. Overall (platelet count > 50 x 10(9)/L) and complete responses (platelet count > 100 x 10(9)/L) were achieved in 21/28 (75%) and 12/28 (43%) patients respectively. The median time to response and time to complete response were 31 and 44 days respectively. After a median follow-up of 11 months (range 3-18), 7/21 (33%) patients relapsed and 3 needed further treatments. In patients with idiopathic thrombocytopenic purpura, lower dose rituximab seems to show similar activity to standard dose.

Transhepatic arterial chemoembolization with oxaliplatin-eluting microspheres (OEM-TACE) for unresectable hepatic tumors.

BACKGROUND: While conventional transhepatic arterial chemoembolization (TACE) is accepted worldwide as an effective treatment for patients with unresectable hepatocellular carcinoma (HCC), its use in other hepatic tumors is not supported by randomized studies. Preliminary results have shown that new drug-eluting microspheres (DEM) seem to optimize TACE procedures. The aim of this study was to evaluate the capability of HepaSphere to load oxaliplatin and their pharmacokinetic outcome. The feasibility and safety of treatment with oxaliplatin-eluting microspheres (OEM-TACE) was also evaluated in patients with unresectable liver metastasis of colorectal cancer and unresectable intrahepatic cholangiocarcinoma. PATIENTS AND METHODS: An inductively coupled plasma mass spectrometer (ICP-MS) was used to quantify the oxaliplatin bound to microspheres and the oxaliplatin in liver biopsies. Fifteen patients (8 with colorectal carcinoma liver metastases, 7 with intrahepatic cholangiocarcinoma) were treated with 27 sessions of OEM-TACE. RESULTS: The data suggested that the microspheres can bind oxaliplatin entirely. The pharmacokinetic parameters were significantly different between the OEM-TACE patients and a control group of patients treated with oxaliplatin chemotherapy. The mean oxaliplatin concentration within the tumor was twenty-times higher than the extratumoral liver concentration in the OEM-TACE patients. According to response evaluating criteria in solid tumors (RECIST), stable disease was observed in 8 out of the 15 patients (53.3%), a partial response in 2 (13.3%) and intrahepatic or extrahepatic tumor progression in 5 out of the 15 patients (33.3%). No major adverse event (AE G3/4) occurred. CONCLUSION: TACE with oxaliplatin-loaded microspheres is a safe and feasible treatment without major adverse events and with a favorable pharmacokinetic profile.

Development and evaluation of an immunoassay for the monitoring of the anti-HIV drug amprenavir.

An assay for routine therapeutic drug monitoring of anti-HIV HAART drugs in clinical use is highly desirable, in order to rapidly measure the pharmacokinetic parameters on single patients. We have started a project to develop a panel of enzyme-linked immunosorbent assays (ELISA) for the whole set of HAART drugs, and the development, performance and evaluation of the assay for amprenavir is described here. A diazo conjugate of amprenavir has been used in order to raise polyclonal anti-amprenavir antibodies in rabbits. Antisera have been used to set up a quantitative and rapid competitive assay. Plasma samples are simply diluted in the assay buffer after thermal inactivation, before running the assay. The assay allows the detection of amprenavir in the quantification range 400-5000 ng/ml, in a diluted plasma sample. The assay has been compared with an HPLC reference technique, on 27 samples from treated patients. Within the quantification range, the ELISA data are well correlated with the HPLC results by a regression line close to the identity, and a Bland-Altman analysis shows the agreement between the two methods.

Plasma alemtuzumab levels in patients with chronic lymphocytic leukemia treated with alemtuzumab combined with chemotherapy reflect the efficacy of the treatment: a hypothesis.

In the HOVON68 trial comparing subcutaneous low-dose alemtuzumab (LD-A) used together with fludarabine (F) and cyclophosphamide (C) with FC alone in high-risk chronic lymphocytic leukemia (CLL), LD-AFC resulted in significantly more clinical and molecular responses than FC, but also in more opportunistic infections. In a subgroup analysis of alemtuzumab trough levels during treatment by a sensitive enzyme-linked immunosorbent assay (ELISA) method, detectable levels were found in 4/6 complete and 0/3 partial responders. A relationship between alemtuzumab plasma levels, response and duration of lymphocytopenia was evident. We hypothesize that following combination therapy, the response may not be a function of the alemtuzumab levels, but the opposite, that plasma alemtuzumab levels are a function of the efficacy of the entire treatment, and the fewer leukemic target cells that are remaining, the higher are the levels of plasma alemtuzumab. This concept may well provide a guide for alemtuzumab dosage in future trials.

Alemtuzumab as consolidation after a response to fludarabine is effective in purging residual disease in patients with chronic lymphocytic leukemia.

PURPOSE: Treatment with alemtuzumab has resulted in negative responses for minimal residual disease (MRD) in patients with chronic lymphocytic leukemia (CLL). In a prior analysis we demonstrated that it is possible to achieved MRD negativity, as assessed by polyclonality of immunoglobulin heavy chain after consolidation with alemtuzumab. This phase II study evaluated 34 patients with CLL who received alemtuzumab consolidation in an effort to improve the quality of their response to fludarabine-based induction. Subsequent peripheral blood stem-cell (PBSC) collection and transplantation, tolerability, and pharmacokinetics also were assessed. PATIENTS AND METHODS: Thirty-four patients younger than 65 years who had a clinical response to fludarabine-based induction therapy received alemtuzumab 10 mg subcutaneously three times per week for 6 weeks. PBSCs were collected after mobilization with cytarabine and granulocyte colony-stimulating factor. Blood samples for pharmacokinetics study were taken between days 1 and 31. RESULTS: The complete response rate improved from 35% after fludarabine induction to 79.4% after alemtuzumab consolidation, including 19 patients (56%) who achieved MRD negativity. The most common adverse events were injection-site reactions and fever. Cytomegalovirus reactivation occurred in 18 patients, all of whom were successfully treated with oral ganciclovir. PBSC collection was successful in 24 (92%) of 26 patients, and 18 patients underwent autologous PBSC transplantation. Alemtuzumab plasma concentrations increased gradually during the first 2 weeks and accumulated more rapidly thereafter. CONCLUSION: Subcutaneously administered alemtuzumab was effective, safe, and well tolerated as consolidation therapy in patients with CLL who responded to fludarabine induction therapy. Subsequent PBSCT was feasible thereafter.

Pharmacokinetic behavior of rituximab: a study of different schedules of administration for heterogeneous clinical settings.

This study was designed to report the pharmacokinetic behavior of Rituximab in patients affected with different diseases and treated with different schedules of administration. A low tumor burden was a common feature of all patients (N=48) included in our study, whereas the timing of Rituximab administration varied from weekly (groups 1, 2, 3) to monthly (group 4). Group 1 included patients with follicular lymphoma treated with 4 weekly doses of Rituximab after first-line chemotherapy with CHOP. At the start of Rituximab, patients were in partial or complete clinical response but showed persistence of disease at molecular level (bcl-2-positive) in bone marrow and/or peripheral blood. Patients in group 2 had autoimmune disorders and Rituximab was given to act on B-cells, interfering with their production of autoantibodies. In patients with amyloidosis (group 3), Rituximab was given to kill progenitor B-cells of the small clone terminating in amyloid-producing plasma cells. In groups 2 and 3, the target of monoclonal antibody was a population of small B cells, which make an intrinsic feature of the diseases. Group 4 included patients with relapsed or refractory follicular and mantle cell lymphoma who underwent a salvage program of immunochemotherapy, purging in vivo and autotransplant: the first of the six planned doses of Rituximab was administered after a debulking phase with a third-generation regimen, such as VACOP-B. An enzyme-linked immunoassay (ELISA) developed and validated in our laboratory was used for the pharmacokinetic study. Rituximab disposition was characterized by a 2-exponential decay, with a long elimination half-life of approximately 3 weeks (range, 248-859 hours). The total systemic clearance ranged between 3.1 and 11.9 mL/hr/m. After 4 weekly infusions, Rituximab concentration was approximately 2.5 microg/mL, which is approximately 85% of the steady-state level. Steady-state plasma concentrations of Rituximab were reached after 6 to 8 weekly infusions. The adopted pharmacokinetic model (2-compartment open model) seems to provide the best fit of Rituximab disposition both during and after treatment, even when different schedules of drug administration are used. Because several studies reported an association between response and serum Rituximab concentrations, a treatment based on a pharmacokinetic model may be useful for predicting the desired drug concentration.