scholarly journals Assessment of l-Asparaginase Pharmacodynamics in Mouse Models of Cancer

Metabolites ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 10 ◽  
Author(s):  
Thomas D. Horvath ◽  
Wai Kin Chan ◽  
Michael A. Pontikos ◽  
Leona A. Martin ◽  
Di Du ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Catalytic Efficiency ◽  
Rapid Quenching ◽  
Kinetic Properties ◽  
Treatment Regimens ◽  
Accuracy And Precision ◽  
Single Animal

l-asparaginase (ASNase) is a metabolism-targeted anti-neoplastic agent used to treat acute lymphoblastic leukemia (ALL). ASNase’s anticancer activity results from the enzymatic depletion of asparagine (Asn) and glutamine (Gln), which are converted to aspartic acid (Asp) and glutamic acid (Glu), respectively, in the blood. Unfortunately, accurate assessment of the in vivo pharmacodynamics (PD) of ASNase is challenging because of the following reasons: (i) ASNase is resilient to deactivation; (ii) ASNase catalytic efficiency is very high; and (iii) the PD markers Asn and Gln are depleted ex vivo in blood samples containing ASNase. To address those issues and facilitate longitudinal studies in individual mice for ASNase PD studies, we present here a new LC-MS/MS bioanalytical method that incorporates rapid quenching of ASNase for measurement of Asn, Asp, Gln, and Glu in just 10 µL of whole blood, with limits of detection (s:n ≥ 10:1) estimated to be 2.3, 3.5, 0.8, and 0.5 µM, respectively. We tested the suitability of the method in a 5-day, longitudinal PD study in mice and found the method to be simple to perform with sufficient accuracy and precision for whole blood measurements. Overall, the method increases the density of data that can be acquired from a single animal and will facilitate optimization of novel ASNase treatment regimens and/or the development of new ASNase variants with desired kinetic properties.

scholarly journals The potential of adoptive transfer of γ9δ2 T cells to enhance blinatumomab’s antitumor activity against B-cell malignancy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yun-Hsiang Chen ◽  
Yun Wang ◽  
Cheng-Hao Liao ◽  
Shu-Ching Hsu
Keyword(s):  
T Cells ◽  
Antitumor Activity ◽  
B Cell ◽  
Adoptive Transfer ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Inadequate Response ◽  
Treatment Regimens ◽  
Extramedullary Disease

AbstractBlinatumomab, a bispecific T cell engager (BiTE) antibody targeting CD19 and CD3ε, can redirect T cells toward CD19-positive tumor cells and has been approved to treat relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL). However, chemotherapeutic regimens can severely reduce T cells’ number and cytotoxic function, leading to an inadequate response to blinatumomab treatment in patients. In addition, it was reported that a substantial portion of R/R B-ALL patients failing blinatumomab treatment had the extramedullary disease, indicating the poor ability of blinatumomab in treating extramedullary disease. In this study, we investigated whether the adoptive transfer of ex vivo expanded γ9δ2 T cells could act as the effector of blinatumomab to enhance blinatumomab’s antitumor activity against B-cell malignancies in vivo. Repeated infusion of blinatumomab and human γ9δ2 T cells led to more prolonged survival than that of blinatumomab or human γ9δ2 T cells alone in the mice xenografted with Raji cells. Furthermore, adoptive transfer of γ9δ2 T cells reduced tumor mass outside the bone marrow, indicating the potential of γ9δ2 T cells to eradicate the extramedullary disease. Our results suggest that the addition of γ9δ2 T cells to the blinatumomab treatment regimens could be an effective approach to enhancing blinatumomab’s therapeutic efficacy. The concept of this strategy may also be applied to other antigen-specific BiTE therapies for other malignancies.

Adenosine Diphosphate (ADP)-Induced ⍺-Granules Release from Platelets of Native Whole Blood Is Reduced by Ticlopidine but Not by Aspirin or Dipyridamole

1986 ◽  
Vol 56 (02) ◽  
pp. 147-150 ◽  
Author(s):  
V Pengo ◽  
M Boschello ◽  
A Marzari ◽  
M Baca ◽  
L Schivazappa ◽  
...  
Keyword(s):  
Whole Blood ◽  
Light Transmission ◽  
Ex Vivo ◽  
Early Stage ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Dose Dependent ◽  
Plateletderived Growth Factor ◽  
Platelet Shape

SummaryA brief contact between native whole blood and ADP promotes a dose-dependent release of platelet a-granules without a fall in the platelet number. We assessed the “ex vivo” effect of three widely used antiplatelet drugs, aspirin dipyridamole and ticlopidine, on this system. Aspirin (a single 800 mg dose) and dipyridamole (300 mg/die for four days) had no effect, while ticlopidine (500 mg/die for four days) significantly reduced the a-granules release for an ADP stimulation of 0.4 (p <0.02), 1.2 (p <0.01) and 2 pM (p <0.01). No drug, however, completeley inhibits this early stage of platelet activation. The platelet release of α-granules may be related to platelet shape change of the light transmission aggregometer and may be important “in vivo” by enhancing platelet adhesiveness and by liberating the plateletderived growth factor.

Effect of inhibition of 5-lipoxygenase metabolism of arachidonic acid on response to endotoxemia in sheep

1988 ◽  
Vol 65 (3) ◽  
pp. 1351-1359 ◽  
Author(s):  
J. W. Coggeshall ◽  
B. W. Christman ◽  
P. L. Lefferts ◽  
W. E. Serafin ◽  
I. A. Blair ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Whole Blood ◽  
Ex Vivo ◽  
Calcium Ionophore ◽  
Lung Mechanics ◽  
Gas Chromatography Mass Spectrometry ◽  
Lipoxygenase Inhibitor ◽  
Pulmonary Hemodynamics ◽  
Lung Lymph

We studied the effects of a 5-lipoxygenase inhibitor, L-651,192, on the pulmonary dysfunction caused by endotoxemia in chronically instrumented unanesthetized sheep. The efficacy and selectivity of L-651,392 were tested by measuring in vivo production of leukotriene B4 (LTB4) and cyclooxygenase products of arachidonic acid after endotoxemia before and after pretreatment with L-651,392 and ex vivo from granulocytes and whole blood stimulated with calcium ionophore from sheep before and 24 h after pretreatment with L-651,392. A novel assay for LTB4 by high-performance liquid chromatography/gas chromatography/mass spectrometry techniques was developed as a measure of 5-lipoxygenase metabolism of arachidonic acid. L-651,392 proved to be an effective in vivo 5-lipoxygenase inhibitor in sheep. L-651,392 blocked the increase in LTB4 observed in lung lymph after endotoxemia in vivo in sheep as well as inhibited by 80% the ex vivo production of LTB4 by granulocytes removed from sheep treated 24 h earlier with L-651,392. Although L-651,392 blocked the increase in cyclooxygenase products of arachidonic acid observed in lung lymph after endotoxemia in vivo in sheep, the drug probably did not function directly as a cyclooxygenase inhibitor. L-651,392 did not attenuate the ex vivo production of thromboxane B2 by whole blood from sheep treated 24 h earlier with the drug. L-651,392 attenuated the alterations in pulmonary hemodynamics, lung mechanics, oxygenation, and lung fluid and solute exchange observed after endotoxemia in sheep. We speculate that 5-lipoxygenase products are a major stimulus for cyclooxygenase metabolism of arachidonic acid after endotoxemia in sheep.

scholarly journals Lipolysis generates platelet dysfunction after in vivo heparin administration

2002 ◽  
Vol 103 (4) ◽  
pp. 433-440 ◽  
Author(s):  
Elijah W. MURIITHI ◽  
Philip R. BELCHER ◽  
Stephen P. DAY ◽  
Mubarak A. CHAUDHRY ◽  
Muriel J. CASLAKE ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Cardiopulmonary Bypass ◽  
Lipoprotein Lipase ◽  
Whole Blood ◽  
Ex Vivo ◽  
Hepatic Lipase ◽  
Platelet Factor 4 ◽  
Platelet Factor

Heparin, when administered to patients undergoing operations using cardiopulmonary bypass, induces plasma changes that gradually impair platelet macroaggregation, but heparinization of whole blood in vitro does not have this effect. The plasma changes induced by heparin in vivo continue to progress in whole blood ex vivo. Heparin releases several endothelial proteins, including lipoprotein lipase, hepatic lipase, platelet factor-4 and superoxide dismutase. These enzymes, which remain active in plasma ex vivo, may impair platelet macroaggregation after in vivo heparinization and during cardiopulmonary bypass. In the present study, proteins were added in vitro to hirudin (200units·ml-1)-anticoagulated blood from healthy volunteers, and the platelet macroaggregatory responses to ex vivo stimulation with collagen (0.6μg·ml-1) were assessed by whole-blood impedance aggregometry. Over a 4h period, human lipoprotein lipase and human hepatic lipase reduced the platelet macroaggregatory response from 17.0±2.3 to 1.5±1.3 and 1.2±0.6Ω respectively (means±S.D.) (both P<0.01; n = 6). Other lipoprotein lipases also impaired platelet macroaggregation, but platelet factor-4 and superoxide dismutase did not. Platelet macroaggregation showed an inverse linear correlation with plasma concentrations of non-esterified fatty acids (r2 = 0.69; two-sided P<0.0001; n = 8), suggesting that heparin-induced lipolysis inhibits platelet macroaggregation. Lipoprotein degradation products may cause this inhibition by interfering with eicosanoids and other lipid mediators of metabolism.

Treatment with Bortezomib Overcomes Resistance to Imatinib in Ph-Leukemia Quiescent Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1426-1426
Author(s):  
Yachiyo Kuwatsuka ◽  
Yosuke Minami ◽  
Ryohei Tanizaki ◽  
Miho Minami ◽  
Akihiro Abe ◽  
...  
Keyword(s):  
Cell Death ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Leukemia Cells ◽  
Imatinib Treatment ◽  
Spleen Cells ◽  
Quiescent Cells ◽  
Nog Mice

Abstract Abstract 1426 Poster Board I-449 Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. In order to examine mechanisms of drug resistance in LSCs and to seek strategies to overcome the resistance, we used Ph-positive acute lymphoblastic leukemia patient cells serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Recently several publications have suggested that proteasome inhibitors can induce selective cell death in LSCs. Spleen cells derived from leukemic NOG mice were treated ex vivo with imatinib and the proteasome inhibitor, bortezomib and cell viablility (PI-/Annexin-V-) was compared between treated and non-treated cells. After treatment with imatinib, significantly more residual cells were observed in the CD34+CD38- population compared to the CD34+CD38+ or CD34-CD38+ populations. With nM level of bortezomib, substantial cell death was induced in all populations with up-regulation of phospho-p53 (Ser15). Phosphorylation of BCR-ABL and CrkL was completely inhibited in all populations with imatinib treatment, but not with bortezomib treatment. Regarding cell cycle states, a higher percentage of Hoechst-33342low/Pyronin-Ylow cells was observed in the CD34+CD38- population relative to the other populations, suggesting more cells in the G0 state among the CD34+CD38- population. In co-culturing with S17 stromal cells, quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib, while substantial cell death including CD34+ population was induced with nM level of bortezomib. We are also investigating more detailed biomarkers in the cell death and effects of these drugs both on the primitive leukemia cells and normal hematopoietic cells using the in vivo leukemic NOG mice systems. These results imply that resistance to imatinib in Ph-positive leukemia quiescent cells is independent of BCR-ABL phosphorylation and that treatment with bortezomib can overcome the resistance of Ph-positive LSCs. Disclosures Kiyoi: Kyowa Hakko Kirin: Consultancy. Naoe: Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.

In vivo monitoring of JAK/STAT and PI3K/mTOR signal transduction inhibition in pediatric CRLF2-rearranged acute lymphoblastic leukemia (ALL).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 9506-9506
Author(s):  
Sarah Kathleen Tasian ◽  
Shannon L. Maude ◽  
Junior Hall ◽  
Tiffaney Vincent ◽  
Charles Grenfell Mullighan ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Signal Transduction Inhibitors ◽  
Background Therapy ◽  
Pediatric All ◽  
Therapeutic Responses

9506 Background: Therapy intensification for children with B-precursor ALL with high-risk genetic lesions has improved relapse-free survival. CRLF2 rearrangements and JAK2 and IL7RA mutations occur in 10-15% of adult and pediatric ALL patients, most of whom relapse. We and others identified aberrant kinase signatures and perturbed JAK/STAT and PI3K/mTOR signal transduction via in vitro studies of CRLF2-rearranged (CRLF2r) ALLs, suggesting the therapeutic relevance of signal transduction inhibitors (STIs). Our creation of CRLF2r ALL xenograft models has enabled rapid preclinical testing of STIs and measurement of in vivo target inhibition. We hypothesized that inhibition of JAK/STAT and PI3K/mTOR phosphosignaling correlates with therapeutic responses in these models. Methods: NOD/SCID/γc-null (NSG) mice well-engrafted with pediatric ALL samples were treated with the JAK inhibitor ruxolitinib, the mTOR inhibitor sirolimus, or vehicle for 72 hours (for signaling response) or 4 weeks (for therapeutic response). Splenocytes were briefly stimulated ex vivo with thymic stromal lymphopoietin (ligand for CRLF2) and stained with human-specific surface and intracellular phosphoantibodies for multi-parameter phosphoflow cytometry analysis. Results: Ruxolitinib-induced inhibition of phospho (p)-JAK2 and pSTAT5 was most pronounced in non-CRLF2r ALLs with novel JAK2-activating BCR-JAK2 and IL7RA/LNK mutations. Sirolimus potently inhibited pS6 and other PI3K/mTOR pathway phosphoproteins in the CRLF2r r ALLs. PSTAT5 and pS6 inhibition correlated with longer-term ruxolitinib- and sirolimus-induced decreases in ALL cell burden, demonstrating therapeutic responses to STIs. Conclusions: Ruxolitinib inhibited JAK/STAT phosphosignaling and markedly decreased leukemic burden in the JAK2-activating BCR-JAK2 and IL7RA/LNK mutant ALL xenografts. Sirolimus potently inhibited PI3K/mTOR (as well as some JAK/STAT) phosphosignaling and had greater therapeutic efficacy than ruxolitinib in the CRLF2r ALLs. The safety of ruxolitinib and of temsirolimus with cytotoxic chemotherapy are currently being established in Children’s Oncology Group Phase I trials.

scholarly journals In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

Blood ◽  
2012 ◽  
Vol 119 (17) ◽  
pp. 4066-4072 ◽  
Author(s):  
Bethan Psaila ◽  
James B. Bussel ◽  
Matthew D. Linden ◽  
Bracken Babula ◽  
Youfu Li ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Whole Blood ◽  
Immune Thrombocytopenia ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Adenosine Diphosphate ◽  
High Dose ◽  
Platelet Surface

Abstract The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

Evaluation of anti-platelet and anti-thrombotic effects of cilostazol with PFA-100® and Multiplate® whole blood aggregometer in vitro, ex vivo and FeCl3-induced thrombosis models in vivo

2011 ◽  
Vol 127 (6) ◽  
pp. 565-570 ◽  
Author(s):  
Chae-Wook Kim ◽  
Jun-Won Yun ◽  
Il-Hong Bae ◽  
Yang-Hui Park ◽  
Yeon Su Jeong ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo

Contribution of whole blood to the control of plasma asymmetrical dimethylarginine

2006 ◽  
Vol 291 (4) ◽  
pp. H1788-H1796 ◽  
Author(s):  
Scott S. Billecke ◽  
Laura A. Kitzmiller ◽  
Joseph J. Northrup ◽  
Steven E. Whitesall ◽  
Masumi Kimoto ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Dominant Role ◽  
Peptide Bond ◽  
Sprague Dawley ◽  
Asymmetrical Dimethylarginine ◽  
Sprague Dawley Rats ◽  
Endogenous Nitric Oxide ◽  
Nos Inhibitor

The endogenous nitric oxide (NO) synthase (NOS) inhibitor asymmetrical dimethylarginine (ADMA) is elevated in many patients and may contribute to the initiation and progression of their disease. While some mechanistic pathways have been identified, tissue-specific contributions to ADMA control remain unclear. We sought to determine if whole blood (WB) could participate in ADMA control ex vivo. Anesthetized male Sprague-Dawley rats underwent exsanguinations, and WB preparations were incubated at 37°C for 5 h. ADMA and symmetrical dimethylarginine were analyzed by high-pressure liquid chromatography. Incubation of lysed red blood cell (RBC) supernatant yielded a significant decrease in ADMA that was blocked by 4124W, a synthetic inhibitor of dimethylarginine dimethylaminohydrolase, the only reported enzyme to hydrolyze ADMA. Hydrolysis of ADMA was diminished by addition of physiologically relevant concentrations of zinc (i.e., 20 μM). Conversely, when rat WB or WB supernatant was incubated at 37°C, it liberated quantities of free ADMA (1–2 μM) that in vivo would likely have pathological consequences. Addition of arginine methyltransferase inhibitors to these incubations did not reduce ADMA release, indicating no dominant role for active protein methylation during these incubations. This ADMA liberation was significantly reduced by addition of protease inhibitors, indicating a dependence on peptide bond hydrolysis. Total ADMA (protein incorporated plus free) was determined by acid hydrolysis and found to be 43.18 ± 4.79 μM in WB with ∼95% of this in RBCs. These ex vivo data demonstrate the potential of blood to control the NO-NOS system by modulating free ADMA.

Platelet ADP receptors contribute to the initiation of intravascular coagulation

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 594-600 ◽  
Author(s):  
Catherine Leon ◽  
Meike Alex ◽  
Antje Klocke ◽  
Eberhard Morgenstern ◽  
Christine Moosbauer ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Intravascular Coagulation ◽  
Adp Receptors ◽  
Adp Receptor ◽  
Deficient Mice ◽  
Storage Pools

Abstract While the adenosine 5′-diphosphate (ADP) pathway is known to enhance thrombus formation by recruiting platelets and leukocytes to the primary layer of collagen-adhering platelets, its role for the initiation of coagulation has not been revealed. Ex vivo inhibition of the P2Y12 ADP receptor by clopidogrel administration diminished the rapid exposure of tissue factor (TF), the major initiator of coagulation, in conjugates of platelets with leukocytes established by the contact of whole blood with fibrillar collagen. Under in vitro conditions, the P2Y12 and P2Y1 ADP receptors were both found to be implicated in the exposure of TF in collagen-activated whole blood. Immunoelectron-microscopy revealed that collagen elicited the release of TF from its storage pools within the platelets. Functional activation of the intravascular TF was reduced by inhibition of the ADP receptors, partially due to the disruption of the platelet-neutrophil adhesions. Injection of collagen into the venous system of mice increased the number of thrombin-antithrombin complexes, indicative for the formation of thrombin in vivo. In P2Y1-deficient mice, the ability of collagen to enhance the generation of thrombin was impaired. In conclusion, the platelet ADP pathway supports the initiation of intravascular coagulation, which is likely to contribute to the concomitant formation of fibrin at the site of the growing thrombus.

Sign in / Sign up

Export Citation Format

Share Document