Imatinib, dasatinib, and nilotinib are tyrosine kinase inhibitors (TKIs) used as first-line treatment of chronic myeloid leukemia. Therapeutic drug monitoring is important to achieve treatment ...efficacy in the case of imatinib and nilotinib, and to control toxicity in the case of dasatinib. New high-sensitivity methods to monitor those drugs are needed, especially for dasatinib. Thus, a simple method to determine plasma levels of imatinib, dasatinib, and nilotinib for application in clinical practice was developed.
TKIs were eluted with a Poroshell 120 EC-C18 column (2.1 × 75 mm, 2.7 μm) at 0.5 mL/min and 60°C, under gradient conditions through a mobile phase consisting of 4 mmol/L ammonium formate, pH 3.2 (65%), and acetonitrile (35%). TKIs were detected and quantified by liquid chromatography in tandem with mass spectrometry (LC/MS-MS) with positive electrospray ionization and analytes were extracted using solid phase extraction (Versaplate-SCX). Internal standards were isotope-labeled for each analyte.
The method was linear in the range of 2.5-5000 ng/mL for imatinib, 0.75-400 ng/mL for dasatinib, and 2-4000 ng/mL for nilotinib. The validation assays for accuracy and precision, matrix effect, extraction recovery, carryover, and stability of the samples for all the TKIs were appropriate according to regulatory agencies. Furthermore, imatinib plasma samples, stored for 4 years at -80°C were quite stable in approximately half of the samples.
The method enables rapid quantification of TKI concentrations and is being applied to therapeutic drug monitoring to adjust dose and to manage adverse reactions in clinical practice.
The aim of the study is to develop a population pharmacokinetic (PopPK) model and to investigate the influence of CYP3A5/CYP3A4 and ABCB1 single nucleotide polymorphisms (SNPs) on the Tacrolimus PK ...parameters after LCP-Tac formulation in stable adult renal transplant patients. The model was developed, using NONMEM v7.5, from full PK profiles from a clinical study (n = 30) and trough concentrations (C
) from patient follow-up (n = 68). The PK profile of the LCP-Tac formulation was best described by a two-compartment model with linear elimination, parameterized in elimination (CL/F) and distributional (CL
/F) clearances and central compartment (Vc/F) and peripheral compartment (Vp/F) distribution volumes. A time-lagged first-order absorption process was characterized using transit compartment models. According to the structural part of the base model, the LCP-Tac showed an absorption profile characterized by two transit compartments and a mean transit time of 3.02 h. Inter-individual variability was associated with CL/F, Vc/F, and Vp/F. Adding inter-occasion variability (IOV) on CL/F caused a statistically significant reduction in the model minimum objective function MOFV (
< 0.001). Genetic polymorphism of CYP3A5 and a cluster of CYP3A4/A5 SNPs statistically significantly influenced Tac CL/F. In conclusion, a PopPK model was successfully developed for LCP-Tac formulation in stable renal transplant patients. CYP3A4/A5 SNPs as a combined cluster including three different phenotypes (high, intermediate, and poor metabolizers) was the most powerful covariate to describe part of the inter-individual variability associated with apparent elimination clearance. Considering this covariate in the initial dose estimation and during the therapeutic drug monitoring (TDM) would probably optimize Tac exposure attainments.
Dasatinib is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which is frequently used to treat chronic myeloid leukemia. Although very effective, patients taking dasatinib often ...display severe adverse effects, including pleural effusions and increased risk of bleeding primarily in the gastrointestinal tract. The actual causes of these side effects are currently undetermined. We hypothesize that endothelial cells (ECs) that line the inner walls of blood vessels and control the traffic to the underlying tissues might be involved.
The effects of TKIs on ECs were studied by various assays, such as real-time cell impedance measurements, live-cell microscopy, wound healing, Western blot, and an
model.
Dasatinib uniquely causes a profound, dose-dependent disorganization of the EC monolayers. Dasatinib promoted the disassembly of cell-cell contacts, altered cell-matrix contacts, and further altered the wound healing. A key observation is that this effect is fully reversible after drug washout. In line with these
observations, intraperitoneal administration of dasatinib to mice caused significant vascular leakage in the intestine. The underlying molecular mechanism of dasatinib-induced reorganization of the actin involves ROCK activation, which increases the amount of the phosphorylation of myosin light chain and consequently activates the non-muscle myosin II.
Our data are consistent with a scenario in which dasatinib triggers a transient increase in vascular leakage that probably contributes to adverse effects such as bleeding diathesis and pleural effusions.
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Dasatinib is a dual SRC/ABL tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML) that is known to have unique immunomodulatory effects. In particular, dasatinib intake typically ...causes lymphocytosis, which has been linked to better clinical response. Since the underlying mechanisms are unknown and SRC family kinases are involved in many cell motility processes, we hypothesized that the movement and migration of lymphocytes is modulated by dasatinib.
Peripheral blood samples from CML patients treated with second-line dasatinib were collected before and 2 h after the first dasatinib intake, and follow-up samples from the same patients 3 and 6 months after the start of therapy. The migratory capacity and phenotype of lymphocytes and differential blood counts before and after drug intake were compared for all study time-points.
We report here for the first time that dasatinib intake is associated with inhibition of peripheral blood T-cell migration toward the homeostatic chemokines CCL19 and CCL21, which control the trafficking toward secondary lymphoid organs, mainly the lymph nodes. Accordingly, the proportion of lymphocytes in blood expressing CCR7, the chemokine receptor for both CCL19 and CCL21, decreased after the intake including both naïve CD45RA+ and central memory CD45RO+ T-cells. Similarly, naïve B-cells diminished with dasatinib. Finally, such changes in the migratory patterns did not occur in those patients whose lymphocyte counts remained unchanged after taking the drug.
We, therefore, conclude that lymphocytosis induced by dasatinib reflects a pronounced redistribution of naïve and memory populations of all lymphocyte subsets including CD4+ and CD8+ T-cells and B-cells.
Chronic lymphocytic leukemia (CLL) with deletions of the
p
53 locus on chromosome 17 and/or refractory to fludarabine chemoimmunotherapy remains a major clinical problem with few therapeutic options. ...Currently, these types of CLL are treated with approaches that do not target the p53 pathway, such as small molecules and monoclonal antibodies (mAb). We have previously postulated anti-CCR7 mAb therapy as a novel CLL treatment. In the present study, we evaluated the in vitro efficacy of anti-CCR7 mAb as a single agent in CLL patients with high-risk cytogenetics and/or refractory to fludarabine, by measuring CCR7 surface expression and complement-dependent cytotoxicity. Our results demonstrate that CCR7 is highly expressed in challenging and heavily treated CLL patients. In addition, the complement-mediated mechanism of action of this mAb effectively eradicates CLL cells while sparing subsets of T cells in these patients. Moreover, this mAb outperformed the activity of alemtuzumab, the mAb with the highest efficacy in these groups. Finally, in vitro activity was also demonstrated in patients with a disease refractory to both fludarabine and alemtuzumab, and patients harboring 11q22 deletion. Our results propose that anti-CCR7 mAb is an effective and promising future treatment in high-risk CLL.
Background and Objective
The gold standard treatment of established cytomegalovirus infection or prevention in solid organ transplantation is the intravenous administration of ganciclovir (GCV) or ...oral administration of valganciclovir (VGCV), both adjusted to the renal function. In both instances, there is a high interindividual pharmacokinetic variability, mainly owing to the wide range of variation of both the renal function and body weight. Therefore, accurate estimation of the renal function is crucial for GCV/VGCV dose optimization. This study aimed to compare three different formulas for estimating the renal function in solid organ transplantation patients with cytomegalovirus infection, for individualizing antiviral therapy with GCV/VGCV, using a population approach.
Methods
A population pharmacokinetic analysis was performed using NONMEM 7.4. A total of 650 plasma concentrations obtained after intravenous GCV and oral VGCV administrations were analyzed, from intensive and sparse sampling designs. Three different population pharmacokinetic models were built with the renal function given by Cockcroft–Gault, Modification of Diet in Renal Disease, or Chronic Kidney Disease EPIdemiology Collaboration (CKD-EPI) formulas. Pharmacokinetic parameters were allometrically scaled to body weight.
Results
The CKD-EPI formula was identified as the best predictor of between-patient variability in GCV clearance. Internal and external validation techniques showed that the CKD-EPI model had better stability and performed better compared with the others.
Conclusions
The model based on the more accurate estimation of the renal function with the CKD-EPI formula and body weight as a size metric most used in the clinical practice can refine initial dose recommendations and contribute to GCV and VGCV dose individualization when required in the prevention or treatment of cytomegalovirus infection in solid organ transplantation patients.
Graphical Abstract
The aim of the study is to develop a population pharmacokinetic (PopPK) model and to investigate the influence of CYP3A5/CYP3A4 and ABCB1 single nucleotide polymorphisms (SNPs) on the Tacrolimus PK ...parameters after LCP-Tac formulation in stable adult renal transplant patients. The model was developed, using NONMEM v7.5, from full PK profiles from a clinical study (n = 30) and trough concentrations (Csub.0) from patient follow-up (n = 68). The PK profile of the LCP-Tac formulation was best described by a two-compartment model with linear elimination, parameterized in elimination (CL/F) and distributional (CLsub.D/F) clearances and central compartment (Vc/F) and peripheral compartment (Vp/F) distribution volumes. A time-lagged first-order absorption process was characterized using transit compartment models. According to the structural part of the base model, the LCP-Tac showed an absorption profile characterized by two transit compartments and a mean transit time of 3.02 h. Inter-individual variability was associated with CL/F, Vc/F, and Vp/F. Adding inter-occasion variability (IOV) on CL/F caused a statistically significant reduction in the model minimum objective function MOFV (p < 0.001). Genetic polymorphism of CYP3A5 and a cluster of CYP3A4/A5 SNPs statistically significantly influenced Tac CL/F. In conclusion, a PopPK model was successfully developed for LCP-Tac formulation in stable renal transplant patients. CYP3A4/A5 SNPs as a combined cluster including three different phenotypes (high, intermediate, and poor metabolizers) was the most powerful covariate to describe part of the inter-individual variability associated with apparent elimination clearance. Considering this covariate in the initial dose estimation and during the therapeutic drug monitoring (TDM) would probably optimize Tac exposure attainments.
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In patients with so called "late suboptimal responses" (patient with complete cytogenetic response (CCyR) without major molecular response (MMR) after 18 months of imatinib) , the role of dasatinib ...has not been evaluated. Dasatinib has unique immunomodulatory effects especially on the proliferation and activation of T- and NK-cells. Yet, how dasatinib affects the migration of lymphocytes is unknown. DASAPOST is the first clinical trial evaluating efficacy and safety of dasatinib in patients with late suboptimal response (now considered as ELN2013 as warning). Another aim is to correlate new immunological aspects related to dasatinib and its possible correlation with responses.
We are presenting results of first 18 patients enrolled in the phase II DASAPOST study (NCT01802450). Main inclusion criteria were patients treated with late suboptimal response by ELN09 (CCyR without MMR after 18 months of treatment. Sokal risk groups were (L/I/H) 22.5%, 50% and 22.5%. All BCR-ABL/ABL (IS) measurements were centralized in an EUTOS laboratory. An exhaustive lymphocyte migration study was done, including immunophenotipying pre and post samples (CD 45, CD3,CD8, CD16, CXCR3, CXCR4, CD56 and CCR7), migration assay (chemokines CXCL10, CCL19+CCL21 and CXCL12) and CXCL10 plasma concentration measured by ELISA.
- Clinical: Median follow up was 288 days (100-380). Three out of 18 (16%) patients had discontinued dasatinib due to side effects (pancreatitis, pleural effusion and low grade, persistant side effects (fever, arthralgias, anemia and astenia). All patients have been evaluated at 3 months, 17 at 6 months and 11 at 12 months. Cumulative incidences by ITT of MMR by 3 and 6 months were 50% and 81%. Cumulative incidences by ITT of MR4.5 by 3 and 6 months were 18% and 25%, respectively.
- Inmunological: Dasatinib intake induced a significant increase of NK-cells and decrease of percentage of T-cells. Further, it increased CD8+ T cells, while reducing the proportion of CD4+ T-cells among the total T-cells. With the first dose of dasatinib (to),the percentage of CCR7 was lower in CD4+ and CD8+ T-cells in the post-samples. Lymphocyte migration was studied with transwell assays. At t0, post-samples showed a reduced migratory capacity towards the chemokines CCL19 and CCL21 in both CD4+ and CD8+ T-cell subsets. Patients were classified as mobilizers (n=14) or non-mobilizers (n=3) depending on whether they experienced an increase in the absolute lymphocyte counts after the first intake of dasatinib or not, showing different lymphocyte distribution and migratory capacity. In order to study the long term effects of dasatinib, we calculated the fold change (FC) of absolute lymphocyte counts pre- and post-dasatinib intake. Patients were divided into two groups based on whether in the 3 months samples (t3) had a higher ("increase group") or a lower ("decrease group") FC compared with t0. The migratory capacity of these two groups was studied in basal conditions and towards CCL19+CCL21 or CXCL10. We found no differences in basal migration in the "increase "group, while, the basal migration in the "decrease" group was quite promoted at t0 and t3. Further, migration towards CCL19+21 in post-samples is even more inhibited in "increase" patients at t3, whereas in the "decrease" patients the inhibition is diminished. The patients were divided into two groups based on the achievement of MMR at t3. At t0 both patient groups had similar migratory capacity, however, at t3, responders maintained significantly impaired migratory capacity to CCL19+21, compared with non-responders.
Our study shows, for the first time to our knowledge, that in patients treated with Imatinib and with late warning responses, switch to Dasatinib induced MMR in 83% of the patients, although 16% discontinued treatment because of toxicity. We reported for the first time that dasatinib has significant effects on lymphocyte migration, and these are associated with early response.
García-Gutierrez:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Casado:Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Sánchez-Guijo:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau. Boque:Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria. Muñoz-Calleja:BMS: Research Funding. Steegmann:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Ariad: Consultancy, Honoraria, Research Funding, Speakers Bureau.
Background. Cytomegalovirus (CMV) is a widespread persistent β–herpesvirus, which can cause severe complications during primary infection or reactivation in immunocompromised patients, such as after ...allogeneic stem cell transplantation (alloSCT). Another major complication associated with alloSCT is graft-versus-host disease (GVHD). The pathogenesis of GVHD involves migration of the transplanted donor naïve T-cells into the secondary lymphoid organs in the recipient, which is mainly steered by CD62L and CCR7. As these homing molecules have been associated with both acute GVHD (aGVHD) and chronic GVHD (cGVHD), we studied whether the CMV serostatus of the donor affects the lymphoid composition of the graft product and whether this phenotype can predict CMV reactivation and GVHD.
Methods. This single-center study included 77 donor-recipient pairs who underwent alloSCT. 64 pairs were HLA identical, 12 had 1 mismatch and 3 had 2 mismatch. 36 donors were related to their recipients. All recipients were followed at least for 100 (aGVHD) or 360 days (cGVHD) after transplantation. 43 donors were CMV-seropositive (CMVpos) and 34 were CMV-seronegative (CMVneg). 62 recipients were CMVpos, and 32 of them developed CMV reactivation, 25 aGVHD and 30 cGVHD. Samples from the graft product (donor) were phenotyped by flow cytometry (CD45, CD3, CD8, CD4, CD62L, CCR7) and both frequency (freq) and absolute number (abs) of each T-cell subpopulation were analyzed.
Results. When the donors were divided based on their CMV serostatus, we observed that the grafts from CMVpos donors had a lower freq of naïve (CCR7+CD62L+) CD4+ T-cells (of lymphocytes p=0.06, of CD3 p=0.06, of CD4 p=0.07) and naïve CD8+ T-cells (of leukocytes p=0.03, of lymphocytes p=0.041, of CD3 p=0.011, of CD8 p=0.012) compared to CMVneg donors. Further, the abs of transplanted naïve CD8+ T-cells was significantly lower in the grafts from CMVpos donors (p=0.048). No differences were observed in T-cells (CD3+, CD4+, CD8+).
We next studied if the CMV-serostatus and T-cell phenotype of the graft associates with GVHD. CMVpos donors whose recipients developed aGVHD had higher abs (p=0.05) and freq of naïve CD8+ T-cells (of lymphocytes p=0.08, of CD3 p=0.08, of CD8 p=0.11) compared to those without aGVHD. The same trend was observed with abs (p=0.11) and freq of CCR7+CD4+ T-cells (of leukocytes p=0.15). Similarly, those CMVpos donors whose recipients developed cGVHD had higher abs (p=0.05) and freq of CCR7+CD8+ T-cells (of leukocytes p=0.03, of lymphocytes p=0.06). Further, cGVHD patients who received the transplant from CMVpos donors were infused with a higher freq of CD3+ (of leukocytes p=0.03) and CD4+ T cells (of leukocytes p=0.04) than patients who received a graft from CMVpos donors but did not develop cGVHD. In contrast, CMVneg donors who developed aGVHD had a higher freq of CD3+ (p=0.018) and CD4+ T-cells (p=0.09), whereas no differences were seen in the T-cell subpopulations. Conversely, the abs (p=0.08) and freq of CCR7+CD4+ T-cells (of leukocytes p=0.11) were higher in those who later developed cGVHD.
To study whether the graft lymphoid composition can be used to predict CMV reactivation, we analyzed the lymphoid composition in the graft product of those donors (both CMVpos and CMVneg) whose recipients were CMV seropositive but did not develop any form of GVHD (to avoid the influence of GVHD in the reactivation of CMV). Despite the low number of patients (CMV reactivation n=9, and no CMV reactivation n=13), we observed trends of higher portion of CD4+ T-cells (p=0.09 of lymphocytes, of CD3 p=0.20) and CCR7+CD4+ T-cells (of lymphocytes p=0.18, of CD4 p=0.16) in those grafts that were transplanted into CMV seropositive recipients who did not reactivate CMV.
Conclusions. CMVpos donors whose recipient developed either aGVHD or cGVHD had a higher abs and freq of naïve CD8+ T-cells, which was not seen with CMVneg donors. This suggests that seropositivity sets the abs and freq of CD8 subpopulations near to a decisive cutoff for the development of GVHD. Conversely, other factors influences the development of GVHD in those patients whose donors were seronegative. In other words, seropositivity of the donor affects the graft composition and thus the risk of GVHD. Finally, our data indicate that a higher proportion of naïve or central memory CCR7+ CD4+ T cells in the donor graft could prevent CMV reactivation suggesting that graft composition affects also CMV reactivation.
No relevant conflicts of interest to declare.
Dasatinib is a second-generation tyrosine kinase inhibitor (TKI), which is used successfully in the treatment of chronic myeloid leukemia (CML). Dasatinib has two unique features when compared to ...other TKIs (imatinib, nilotinib); first, dasatinib has a significantly shorter half-life in the plasma and second, dasatinib inhibits a wider spectrum of kinases, including several kinases known to be important in the function of the immune system (src, tec, and syk families), which are not affected by the other TKIs. Interestingly, it has been recently shown that both short-term exposure to dasatinib in vivo and long-term treatment with dasatinib improves NK-cell cytotoxicity, however, the mechanisms are not known.
To study the improved cytotoxicity observed in dasatinib-treated patients, we aimed to perform a complete NK-cell phenotyping in these patients. Finally, our goal is to correlate NK-cell phenotype with NK-cell cytotoxicity, and to study the possible correlation between phenotypical changes, NK-cell function and clinical outcome.
This study included 19 dasatinib-treated (DA) CML patients, both first-line (n=7) and second-line (n=12). To investigate the specificity of the immunomodulatory effects of dasatinib, a control group of 9 CML patients treated with imatinib (IM) and another group of 12 healthy donors (HD) were included. Peripheral blood samples obtained before the patients took their daily drug dose were phenotyped with a comprehensive 8-color flow cytometry panel (total 32 antibodies, table 1).
To study the correlation between phenotypical changes and NK-cell cytotoxicity, we performed a standard CD107 degranulation assay. Mononuclear cells were incubated for 6 hours in the presence of the target cell line K562 and a CD107 antibody. CD107 positive NK-cells were then phenotyped with the same panel of 32 antibodies.
All results are summarized in table 1. In brief, DA- and IM-treated CML patients and HD had equal proportions of NK-cells (CD3negCD56+) of total lymphocytes. Regarding trafficking molecules, NK-cells in both DA- and IM-patients had a lower frequency of the chemokine receptor CCR7 when compared to HD. This suggests a reduction in the NK-cell population that is able to migrate to lymph nodes, and is likely caused by the disease or TKIs in general. Moreover, DA-treatment specifically decreases the expression of the homing molecule CD62L in NK-cells.
In addition, NK-cells in DA-patients, when compared to IM and HD, expressed less CD11b and significantly more often CD11c and HLA-DR, which reproduce the immunophenotypic changes that typically occurs in recently activated NK-cells and has been shown to associate with improved clinical benefits. Conversely, increased expression of CD57 together with a lower frequency of CD27 and CD28 were observed in both groups of patients and were similar to those typically observed in conditions of chronic NK-cell stimulation.
In contrast, DA-patients had a lower frequency of most of the studied NK-receptors (Nkp30, Nkp46, NKG2D, CD94, CD161, KIR2DL1/S1) when compared to IM and HD. This suggests that NK-cells in DA-treated patients have a more mature phenotype, which is caused by the treatment.
NK-cells in TKI-treated CML patients display a mature phenotype, which is often observed after chronic stimulation suggesting that TKIs have immunomodulatory effects on NK-cells or the disease itself causes the changes. Interestingly, NK-cells in DA-treated patients express a highly differentiated phenotype characterized by high expression of CD57, and decreased expression of Nkp30, Nkp46 and CD161. Similar changes were not seen in IM-patients or HD. It is possible that NK-cells expressing this phenotype might also represent those NK-cells that have previously been driven into clonal expansion by encounters with pathogens because of the specific immunomodulatory effects of dasatinib. This phenotype of highly mature NK-cells, which is associated with high cytolytic potential, could be responsible for the previously described enhanced NK-cytotoxicity caused by dasatinib. In accordance, our preliminary results suggest that these unique phenotypic changes observed in DA-treated patients correlates with the cytotoxic potential. Studies to correlate these results with therapy outcome are ongoing.
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Garcia:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Research Funding. Steegmann:Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau.
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