In Vitro and In Vivo Inhibition of the T315I Mutant BCR/ABL Kinase.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2182-2182 ◽  
Author(s):  
Hong Zhu ◽  
Ehab Hanna ◽  
Dan Lohse ◽  
Silva Stoughton ◽  
Betty Tam ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Pharmacokinetic Profile ◽  
Atp Binding Site ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Medical Need ◽  
T315i Mutation ◽  
Mutant Kinase

Abstract The T315I point mutation situated at the ATP-binding site of BCR/ABL tyrosine kinase remains to be the major unmet medical need in treating CML patients. None of the marketed CML drugs, including Imatinib or the recently approved Dasatinib, are able to inhibit this clinically most prevalent mutant of BCR/ABL kinase. To overcome the T315I mutation-presented hurdle, a novel small molecule BCR/ABL inhibitor, TG101223, has been synthesized and characterized in vitro and in vivo. Enzymatic activity of T315I mutant kinase and proliferation of BaF3:BCR/ABLT315I cells were inhibited with an IC50 of 50 nM and EC50 of 500 nM respectively. However, little effect of TG101223 was observed on the proliferation of the control BaF3 cells (IC50≥5,000 nM). Consistent with these observations, 24 hour treatment with 2,000 nM TG101223 induced DNA fragmentation in BCR/ABLT315I cells, but not in the control BaF3 cells. Furthermore, caspase-3 activation was detected in BCR/ABLT315I cells by an in-cell caspase assay following 4 h treatment with 2,000 nM TG101223. The above results strongly suggest that TG101223 inhibits BCR/ABLT315I-mediated cell proliferation and survival. In contrast, Imatinib did not inhibit AblT315I enzyme nor BCR/ABLT315I cell proliferation. The kinase selectivity of TG101223 was determined using a commercial enzyme assay with a panel of 49 phylogentically diverse protein kinases. Only Abl, Flt3 and the mutants of those two kinases were inhibited by more than 80% with 500 nM TG101223. Importantly, TG101223 demonstrated a desirable pharmacokinetic profile in mouse (oral bioavailability: 40%, clearance: 23 mL/min/kg and T½: 5 h). The tumorogenicity of BCR/AblT315I cells were confirmed by the observation that tail vein infusion with BCR/ABLT315I cells induced splenomegaly in SCID mice and death by day 14. To assess the in vivo activities of TG101223, mice were dosed orally once with TG101223 on day 13, followed by harvesting of their spleens 7 hours after dosing for Western blot-based protein phosphorylation analysis. A significant induction in phosphorylation of both CrkL and BCR/ABL was observed in the spleens of T315I cell recipient mice, supporting the notion that splenomegaly is a result of the infiltration of proliferating BCR/ABLT315I cells. The above induction in spleen CrkL and BCR/ABL phosphorylation was inhibited by TG101223. These data suggest that TG101223 is a promising candidate for treating the drug resistant CML patients.

CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1224-1224
Author(s):  
Jerry C. Cheng ◽  
Dejah Judelson ◽  
Kentaro Kinjo ◽  
Jenny Chang ◽  
Elliot Landaw ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Leukemia Cells ◽  
Mouse Bone Marrow ◽  
T315i Mutation

Abstract The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products

1985 ◽  
Vol 5 (11) ◽  
pp. 3116-3123
Author(s):  
J B Konopka ◽  
O N Witte
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Myelogenous Leukemia ◽  
Gene Products ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Assay Conditions

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.

Dual Src/Abl Kinase Targeted Inhibition in Myeloma Microenvironment Promotes Myeloma Cell Apoptosis Both in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2813-2813
Author(s):  
Karthik Ramasamy ◽  
Lee Macpherson ◽  
Ghulam J Mufti ◽  
Stephen Schey ◽  
Yolanda Calle
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Stromal Cells ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Abl Kinase ◽  
Osteoclast Function

Abstract Abstract 2813 Poster Board II-789 Osteoclast, in addition to eroding the bone resulting in lytic lesions, enhances plasma cell proliferation and survival via direct cell to cell contact. Src family protein tyrosine kinases (SFKs) and c-Abl kinase play important role downstream of integrin adhesion receptors, and regulate the cytoskeletal organisation, cell motility and gene expression in response to cell adhesion. We hypothesised targeting SFKs and Abl kinase with the small molecule tyrosine kinase inhibitor Dasatinib has potential to reduce adhesion of plasma cells to ECM proteins in the bone marrow and modify the microenvironment by inhibiting osteoclast function, specifically bone resorption. As a result, myeloma cells could be sensitised to drugs with cytotoxic properties such as dexamethasone. Osteoclasts were generated from primary bone marrow mononuclear cells of myeloma and MGUS patients (n=10). Using Immunofluorescence, we found that Dasatinib 100nM but not dexamethasone inhibited osteoclastogenesis and disrupted the actin cytoskeletal organisation with actin clusters formed in the periphery of the cell. There was absence of actin ring formation at sealing zones which is essential for bone resorption. This effect consistently led to impaired osteoclast function, evidenced by fewer resorption pits formed on rabbit dentine slices on toluidine blue staining. Experiments were repeated ≥ 3 times. In plasma cells, the combination of dexamethasone and Dasatinib synergistically (Calcusyn software) inhibited cell proliferation at clinically relevant concentrations and induced apoptosis of human and murine myeloma cell lines alone and in cocultures with human stromal cells ( p<.001). Dasatinib alone at 200 nM concentration does not inhibit plasma cell proliferation with maximal serum concentration achieved in Phase I CML trials being 180nM. Additionally, Dasatinib and Dexamethasone in combination inhibited secretion of IL-6 but not MIL -1 alpha in stromal cell cocultures. Dasatinib but not dexamethasone significantly inhibited adhesion of myeloma cell lines on Fibronectin despite integrin activation with Magnesium EGTA. This effect was mediated through down regulation of both Src and Abl phosphorylation. Both Dasatinib and Dexamethasone inhibited adhesion of PC on stromal cells and osteoclasts. Taken together, our in vitro results suggest that Dasatinib and dexamethasone could be an effective therapeutic combination with Dasatinib impairing adhesion of plasma cells to the bone marrow microenvironment as well as osteoclast function and resultant bone disease thereby sensitising myeloma cells to the cytotoxic effect of dexamethasone. We have also established that the combination of Dasatinib 75mg/kg and dexamethasone 1mg/kg is not toxic to C57BL/KaLwRij mice. The anti-myeloma efficacy of these drugs alone and in combination is being currently studied. The combination of Dasatinib 100 mg OD days 1-28 and Dexamethasone 20mg OD on Day 1-4, 15-18 has resulted in a partial response (EBMT criteria) in 2 multiply relapsed and steroid refractory myeloma patients without significant toxicity. Serum calcium levels fell commensurate with disease response and we are currently performing experiments to analyse the effect of the drug combination on osteoclast function in vivo. These findings warrant exploring this drug combination in steroid resistant myeloma and patients with extensive skeletal disease prospectively in a phase I/II trial. Disclosures: Off Label Use: Dasatinib is not licensed for Myeloma.

scholarly journals Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products.

1985 ◽  
Vol 5 (11) ◽  
pp. 3116-3123 ◽  
Author(s):  
J B Konopka ◽  
O N Witte
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Myelogenous Leukemia ◽  
Gene Products ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Assay Conditions

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.

Dasatinib Once-Daily Dose Regimen Provides Robust Anti-Leukemic Activity While Avoiding Suppression of T Cell Activation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2198-2198
Author(s):  
Gary L Schieven ◽  
Rosemary Zhang ◽  
Sidney Pitt ◽  
Kelly McGlinchey ◽  
Krista Menard ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Once Daily ◽  
Abl Kinase ◽  
Daily Dose

Abstract Abstract 2198 Poster Board II-175 Dasatinib (SPRYCEL®), a small molecule tyrosine kinase inhibitor is 325-fold more potent against BCR-ABL than imatinib. Targeting BCR-ABL in chronic myeloid leukemia (CML), dasatinib offers the most favorable benefit-risk ratio with the dose regimen of 100-mg once daily (in comparison with 3 other treatment arms: 50 mg BID, 140 mg QD and 70 mg BID. Duration of cytogenetic response and progression-free survival were similar across all 4 arms, but there was significantly less frequent grade 3-4 neutropenia, thrombocytopenia, anemia and pleural effusion in the 100-mg QD arm compared to the other 3 arms combined (Shah et al, J Clin Oncol 26:3204, 2008). The undiminished efficacy of once daily dosing occurs despite the fact that orally administered dasatinib has a pronounced peak-to-trough plasma pharmacokinetics profile and a relatively short half-life (∼3-5 h), which allows for complete recovery of BCR-ABL kinase activity within 8-12 hrs after a daily dose. The clinical efficacy of once daily dasatinib supports the notion that continuous target (BCR-ABL) inhibition is not required for anti-leukemic activity although the truncated exposure may help to ameliorate other side-effects of “on” or “off” targets inhibition. In addition to BCR-ABL, dasatinib potently inhibits SRC-family kinases (SFKs) with an IC50 in the low single digit nM range; SFKs play key roles in T cell activation. Based on continuous exposure studies, it had been suggested that dasatinib may act as an immunomodulator in vivo. We investigated the effects of variation in exposure duration in vivo and in vitro on the anti-leukemic and T cell activation inhibition activities of dasatinib in preclinical models. Methods. Anti-leukemic activity was determined in vitro in K562 and KU812 CML cell lines, and in vivo as xenografts in K562. BCR-ABL kinase activity was monitored with a phospho-specific CRKL antibody. Human T cells were isolated from PBMC by rosetting with sheep red blood cells and stimulated with anti-CD3/CD28 antibodies for 48 h. Cytokines production were measured by ELISA. T cell proliferation was determined at 3 days by 3H-thymidine incorporation. Immunocompetency of dasatinib treated mice were determined using the mouse cardiac allograft model and the in vivo MLR T cell proliferation model. Results. Transient (1-6 h) dasatinib exposure of CML cells that caused >80% inhibition of phospho-CRKL is highly cytotoxic. Degree of cytotoxicity directly correlates with the magnitude of BCR-ABL kinase inhibition. In vivo single dose of 30 mg/kg dasatinib administered IV was highly cytotoxic to K562 xenografts as determined by in vivo-in vitro colony formation assay. Intermittent IV dosing regimens of dasatinib (Q4D or Q7D) were effective against K562 xenografts. Dosing regimen in mice (5 mg/kg, PO) that closely mimic the pharmacokinetics of 100 mg oral dose in human was equally efficacious as administering the same dose in 2 split doses (BID, 2.5 mg/kg, PO). In terms of effects on T cell activation, a linear relationship was observed between serum concentration and in vitro T cell proliferation IC50 values. Modeling the human PK profile, delayed dasatinib treatment of T cells after T cell stimulation in vitro led to a time dependent decrease in potency as measured by both IC50 and Emax values. Comparison of serum adjusted IC50 values from these studies to the human PK profile suggests that dasatinib at the approved 100-mg once-daily dose would permit T cell activation on a daily basis. A similar pattern was observed in preclinical in vivo models. Dasatinib was found to be completely protective in a mouse model of cardiac allograft rejection at a dose of 25 mg BID, whereas a dose of 15 mg BID was not protective. In the MLR model, dasatinib inhibits T cell proliferation at 50 mg/kg but at the clinically relevant dose of 5 mg/kg was completely devoid of T cell inhibitory effects. Taken together, these results suggest that dasatinib may be able to provide anti-leukemic activity while avoiding suppression of T cell activation at clinically relevant doses. Disclosures: Schieven: Bristol-Myers Squibb Co: Employment. Zhang:Bristol-Myers Squibb Co: Employment. Pitt:Bristol-Myers Squibb Co: Employment. McGlinchey:Bristol-Myers Squibb Co: Employment. Menard:Bristol-Myers Squibb Co: Employment. Smykla:Bristol-Myers Squibb Co: Employment. Susulic:Bristol-Myers Squibb Co: Employment. Wen:Bristol-Myers Squibb Co: Employment. Wiebesiek:Bristol-Myers Squibb Co: Employment. Townsend:Bristol-Myers Squibb Co: Employment. Lee:Bristol-Myers Squibb Co: Employment.

A Novel SET Antagonist (OP449) Is Cytotoxic to CML Cells, Including the Highly-Resistant BCR-ABLT315I Mutant, and Demonstrates Enhanced Efficacy in Combination with ABL Tyrosine Kinase Inhibitors,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3757-3757 ◽  
Author(s):  
Anupriya Agarwal ◽  
Ryan MacKenzie ◽  
Jessica Oddo ◽  
Michael P Vitek ◽  
Dale J. Christensen ◽  
...  
Keyword(s):  
K562 Cells ◽  
Pp2a Activity ◽  
Downstream Signaling ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Pp2a Inhibitor

Abstract Abstract 3757 Background: The majority of patients with chronic myeloid leukemia (CML) achieve durable remissions with ABL tyrosine kinase inhibitor (TKI) therapy. However, a subset of CML patients develop resistance to ABL TKIs, either through BCR-ABL kinase-dependent (e.g. kinase domain point mutations) or BCR-ABL kinase-independent (e.g. overexpression of LYN) mechanisms. Recent studies have demonstrated that the presence of BCR-ABL in CML cells result in overexpression of the protein phosphatase 2A (PP2A) inhibitor SET, which in turn prevents PP2A from performing its regulatory role in deactivating signaling proteins through dephosphorylation. Further, loss of PP2A function accounts for increased and sustained BCR-ABL activity in CML blast crisis cells and activation of PP2A via treatment with Forskolin or FTY720 reduces clonogenicity of CML cells, regardless of their sensitivity to ABL TKIs. Therefore, given the central role of PP2A in regulating both BCR-ABL kinase-dependent and kinase-independent downstream signaling, pharmacological activation of PP2A may provide a novel approach to the development of CML therapeutics, particularly for patients with imatinib-resistant CML. Results: Here, we report a novel, specific, cell penetrating peptide known as OP449 that binds to SET and antagonizes SET's inhibition of PP2A. Initial in vitro studies of OP449 in human (K562) and murine (32Dp210) CML cell lines revealed a robust (45%) increase in PP2A activity after treatment with 1μM OP449 when compared to vehicle treated cells. OP449 was more potent than the PP2A activator FTY720 (5μM; 20% increase in PP2A activity compared to vehicle treated control). In comparative cytotoxicity studies, treatment with OP449 consistently resulted in reduced proliferation and increased apoptosis compared to FTY720 in K562 (IC50: 2.5μM vs. 5μM, respectively) and LAMA (IC50: 1.25μM vs. 2.5μM, respectively) cells. Consistent with the role of OP449 as a PP2A activator, its growth inhibitory effects in CML cells were rescued by treatment with okadaic acid, a PP2A inhibitor, indicating that OP449 specifically activates PP2A. Furthermore, the cytotoxic effects of OP449 are more selective for BCR-ABL-positive cells (K562, LAMA, 32Dp210 IC50 range: 1.0 –2.5μM) relative to parental 32D cells (IC50: 5μM). Importantly, TKI-resistant mutant CML cells were also sensitive to OP449 at similar doses (32Dp210T315I cells IC50: 2.5μM). Similarly, in primary CML CD34+ cells, we noted CFU-GM colony formation was reduced by 75% and 98% with 2.5 μM and 5 μM OP449, respectively, while no toxicity was observed in normal human CD34+ cells at concentration of 5 μM. Intriguingly, the combination of OP449 with ABL TKIs (imatinib 1.0μM, nilotinib 50nM, or dasatinib 5nM) further reduced proliferation and increased apoptosis in K562 cells and in primary CD34+ CML cells combination of 2.5 μM OP449 with nilotinib (50nM) reduced colony formation by 98%, which was more than treatment with each drug alone. These data support the potential use of OP449 in combination with ABL TKIs. Mechanistically, the amount of SET protein remained unchanged with OP449 treatment, although levels of CIP2a, another PP2A inhibitor, were reduced significantly. OP449 also inhibited BCR-ABL-mediated downstream signaling, and induced dephosphorylation and degradation of BCR-ABL, STAT5, AKT, ERK, and p38/MAPK in CD34+ CML cells and K562 cells within 6 hr of treatment. Lastly, in vivo toxicity studies did not identify any toxicity of OP449 in non-leukemic mice up to 10mg/kg/day. In a 32Dp210 xenograft model, mice treated with OP449 survived slightly but significantly longer than vehicle-treated mice (p=0.05). In a murine CML bone marrow transplantation model, increasing doses of OP449 correlated with improved survival, reduced WBC count, and reduced GFP+ leukemic cells. Combination treatment of OP449 with nilotinib showed further reduced leukemia burden as compared to monotherapy. Conclusions: Taken together, these data demonstrate that OP449 is selectively cytotoxic to CML cells both in vitro and in vivo by antagonizing SET and increasing PP2A activity, thus restoring PP2A's tumor suppressor function. Furthermore, these data validate SET antagonism as a target for development of new CML therapeutics for treatment of CML patients with resistance to ABL TKIs either alone or in combination with ABL TKIs. Disclosures: Oddo: Oncotide: Employment, Research Funding. Vitek:Oncotide: Employment, Equity Ownership. Christensen:Oncotide: Employment, Equity Ownership. Druker:Bristol-Myers-Squibb: OHSU has clinical trial contracts with Novartis and Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead.; Novartis: OHSU has clinical trial contracts with Novartis and Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead.; MolecularMD: Equity Ownership, verbatim: OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Commi; Cylene: Consultancy; Ariad pharmaceuticals: Consultancy.

Significance of In-Vitro and In-Vivo Correlation in Drug Delivery System

2018 ◽  
Vol 4 (4) ◽  
pp. 523-531
Author(s):  
Hina Mumtaz ◽  
Muhammad Asim Farooq ◽  
Zainab Batool ◽  
Anam Ahsan ◽  
Ashikujaman Syed
Keyword(s):  
Dosage Form ◽  
Pharmaceutical Preparations ◽  
Pharmacokinetic Profile ◽  
In Vitro Dissolution ◽  
Time Saving ◽  
Pharmaceutical Dosage Form ◽  
Short Period ◽  
Form Development

The main purpose of development pharmaceutical dosage form is to find out the in vivo and in vitro behavior of dosage form. This challenge is overcome by implementation of in-vivo and in-vitro correlation. Application of this technique is economical and time saving in dosage form development. It shortens the period of development dosage form as well as improves product quality. IVIVC reduce the experimental study on human because IVIVC involves the in vivo relevant media utilization in vitro specifications. The key goal of IVIVC is to serve as alternate for in vivo bioavailability studies and serve as justification for bio waivers. IVIVC follows the specifications and relevant quality control parameters that lead to improvement in pharmaceutical dosage form development in short period of time. Recently in-vivo in-vitro correlation (IVIVC) has found application to predict the pharmacokinetic behaviour of pharmaceutical preparations. It has emerged as a reliable tool to find the mode of absorption of several dosage forms. It is used to correlate the in-vitro dissolution with in vivo pharmacokinetic profile. IVIVC made use to predict the bioavailability of the drug of particular dosage form. IVIVC is satisfactory for the therapeutic release profile specifications of the formulation. IVIVC model has capability to predict plasma drug concentration from in vitro dissolution media.

Design, Synthesis, and Pharmacokinetic Evaluation of O-Carbamoyl Tizoxanide Prodrugs

2020 ◽  
Vol 16 ◽  
Author(s):  
Xi He ◽  
Wenjun Hu ◽  
Fanhua Meng ◽  
Xingzhou Li
Keyword(s):  
Plasma Concentration ◽  
Broad Spectrum ◽  
Plasma Concentrations ◽  
Antiviral Drug ◽  
Systemic Exposure ◽  
Pharmacokinetic Profile ◽  
Hydroxyl Group ◽  
First Pass

Background: The broad-spectrum antiparasitic drug nitazoxanide (N) has been repositioned as a broad-spectrum antiviral drug. Nitazoxanide’s in vivo antiviral activities are mainly attributed to its metabolitetizoxanide, the deacetylation product of nitazoxanide. In reference to the pharmacokinetic profile of nitazoxanide, we proposed the hypotheses that the low plasma concentrations and the low system exposure of tizoxanide after dosing with nitazoxanide result from significant first pass effects in the liver. It was thought that this may be due to the unstable acyloxy bond of nitazoxanide. Objective: Tizoxanide prodrugs, with the more stable formamyl substituent attached to the hydroxyl group rather than the acetyl group of nitazoxanide, were designed with the thought that they might be more stable in plasma. It was anticipated that these prodrugs might be less affected by the first pass effect, which would improve plasma concentrations and system exposure of tizoxanide. Method: These O-carbamoyl tizoxanide prodrugs were synthesized and evaluated in a mouse model for pharmacokinetic (PK) properties and in an in vitro model for plasma stabilities. Results: The results indicated that the plasma concentration and the systemic exposure of tizoxanide (T) after oral administration of O-carbamoyl tizoxanide prodrugs were much greater than that produced by equimolar dosage of nitazoxanide. It was also found that the plasma concentration and the systemic exposure of tizoxanide glucuronide (TG) were much lower than that produced by nitazoxanide. Conclusion: Further analysis showed that the suitable plasma stability of O-carbamoyl tizoxanide prodrugs is the key factor in maximizing the plasma concentration and the systemic exposure of the active ingredient tizoxanide.

scholarly journals Obesity Potentiates Esophageal Squamous Cell Carcinoma Growth and Invasion by AMPK-YAP Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Jia-Huang Liu ◽  
Qi-Fei Wu ◽  
Jun-Ke Fu ◽  
Xiang-Ming Che ◽  
Hai-Jun Li
Keyword(s):  
Cell Proliferation ◽  
Squamous Cell Carcinoma ◽  
Esophageal Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Nude Mice ◽  
Serum Glucose ◽  
Migration And Invasion ◽  
Endocrine Effect

Obesity could increase the risk of esophageal squamous cell carcinoma (ESCC) and affect its growth and progression, but the mechanical links are unclear. The objective of the study was to explore the impact of obesity on ESCC growth and progression utilizing in vivo trials and cell experiments in vitro. Diet-induced obese and lean nude mice were inoculated with TE-1 cells, then studied for 4 weeks. Serum glucose, insulin, leptin, and visfatin levels were assayed. Sera of nude mice were obtained and then utilized to culture TE-1. MTT, migration and invasion assays, RT-PCR, and Western blotting were used to analyze endocrine effect of obesity on cell proliferation, migration, invasion, and related genes expression of TE-1. Obese nude mice bore larger tumor xenografts than lean animals, and were hyperglycemic and hyperinsulinemic with an elevated level of leptin and visfatin in sera, and also were accompanied by a fatty liver. As for the subcutaneous tumor xenograft model, tumors were more aggressive in obese nude mice than lean animals. Tumor weight correlated positively with mouse body weight, liver weight of mice, serum glucose, HOMA-IR, leptin, and visfatin. Obesity prompted significant TE-1 cell proliferation, migration, and invasion by endocrine mechanisms and impacted target genes. The expression of AMPK and p-AMPK protein decreased significantly ( P < 0.05 ); MMP9, total YAP, p-YAP, and nonphosphorylated YAP protein increased significantly ( P < 0.05 ) in the cells cultured with conditioned media and xenograft tumor from the obese group; the mRNA expression of AMPK decreased significantly ( P < 0.05 ); YAP and MMP9 mRNA expression increased significantly ( P < 0.05 ) in the cells exposed to conditioned media from the obese group. In conclusion, the altered adipokine milieu and metabolites in the context of obesity may promote ESCC growth in vivo; affect proliferation, migration, and invasion of ESCC cells in vitro; and regulate MMP9 and AMPK-YAP signaling pathway through complex effects including the endocrine effect.

scholarly journals Aberrantly high activation of a FoxM1–STMN1 axis contributes to progression and tumorigenesis in FoxM1-driven cancers

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Jun Liu ◽  
Jipeng Li ◽  
Ke Wang ◽  
Haiming Liu ◽  
Jianyong Sun ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Poor Prognosis ◽  
Soft Agar ◽  
Transcriptional Level ◽  
Regulation Mechanism ◽  
Strong Positive Correlation ◽  
Cell Viability Assay ◽  
High Level

AbstractFork-head box protein M1 (FoxM1) is a transcriptional factor which plays critical roles in cancer development and progression. However, the general regulatory mechanism of FoxM1 is still limited. STMN1 is a microtubule-binding protein which can inhibit the assembly of microtubule dimer or promote depolymerization of microtubules. It was reported as a major responsive factor of paclitaxel resistance for clinical chemotherapy of tumor patients. But the function of abnormally high level of STMN1 and its regulation mechanism in cancer cells remain unclear. In this study, we used public database and tissue microarrays to analyze the expression pattern of FoxM1 and STMN1 and found a strong positive correlation between FoxM1 and STMN1 in multiple types of cancer. Lentivirus-mediated FoxM1/STMN1-knockdown cell lines were established to study the function of FoxM1/STMN1 by performing cell viability assay, plate clone formation assay, soft agar assay in vitro and xenograft mouse model in vivo. Our results showed that FoxM1 promotes cell proliferation by upregulating STMN1. Further ChIP assay showed that FoxM1 upregulates STMN1 in a transcriptional level. Prognostic analysis showed that a high level of FoxM1 and STMN1 is related to poor prognosis in solid tumors. Moreover, a high co-expression of FoxM1 and STMN1 has a more significant correlation with poor prognosis. Our findings suggest that a general FoxM1-STMN1 axis contributes to cell proliferation and tumorigenesis in hepatocellular carcinoma, gastric cancer and colorectal cancer. The combination of FoxM1 and STMN1 can be a more precise biomarker for prognostic prediction.

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