Taurine consistent, regardless of the variation in INR control by the study centers among patients assigned to receive warfarin.58 However, for secondary outcomes such as all vascular events, nonhemorrhagic events, and mortality, the advantages of dabigatran in RE LY were more pronounced in patients with poor INR control than those with good INR control. In subgroup analysis in patients with prior stroke or transient ischemic attack, 150 mg of dabigatran comparatively reduced stroke or systemic embolism compared with warfarin.59 Dabigatran has been approved by FDA for stroke prevention in atrial fibrillation. However, intracerebral hemorrhage is a major concern since there is no effective antagonist for dabigatran. However, PCC has been studied in murine model of ICH associated with dabigatran.60 This study demonstrated strong evidence that PCC effectively disufenton sodium prevents intracerebralhematoma expansion in a murine model. However, the role of PCC in humans was inconsistent with the animal model. Eerenberg et al randomly allocated healthy volunteer who received dabigatran 150 mg twice daily, followed by a single bolus of PCC.
This study concluded that PCC has no influence on the anticoagulation action of dabigatran.12 A cost effective analysis of dabigatran compared HSP with adjusted dose warfarin for preventing ischemic stroke in elderly patients 65 years with nonvalvular atrial fibrillation, based on data from RE LY trial and other relevant publications of anticoagulation suggested that dabigatran is more cost effective compared with warfarin. The incremental costeffectiveness ratio compared with warfarin was $51 229 per QALY for low dose dabigatran and $45 372 per QALY for high dose dabigatran.61 However, there were several limitations of this study. First, event rates were mostly derived from a single randomized clinical trial and extrapolated to a 35 year time frame clinical trials, with approximately 2 years of follow up. Second, the cost of dabigatran was estimated on the basis of pricing in the United Kingdom. Acute coronary syndrome. A phase II dose escalating trial for Dabigatran Etexilate in Patients With Acute Coronary Syndrome was a double blind, placebo controlled study doxorubicin which randomized 1861 patients with acute coronary syndrome and at least 1 cardiovascular risk factor to either placebo or dabigatran at the 1 to 4 dosages twice daily, starting within a few days after acute coronary events and continuing for 6 months. In this study population, 99.2% were on dual antiplatelet therapy.
The primary outcome was the composite of major or clinically relevant minor bleeding during the 6 month treatment period. The study showed that combined treatment of dabigatran and dual antiplatelet therapy was associated with a dose dependent increase in bleeding events for 50 mg, HR 2.17 for 75 mg, HR 3.92 for 110 mg, and HR 4.27 for 150 mg. However, dabigatran significantly reduced coagulation activity and may have benefit to reduce cardiovascular events when added to dual antiplatelet therapy in the doses of 110 to 150 mg twice daily. Among other novel anticoagulation therapies, dabigatran has relatively poor oral bioavailability and needs acidic environment to facilitate absorption. Renal excretion is a major route of drug elimination.
Drug library to have a lower incidence of major bleeding than warfarin, in the event of catastrophic hemorrhage no effective reversal agent exists. Neurosurgeons are likely to encounter this clinical scenario more frequently with the increased use of dabigatran and other similar drugs. Preclinical and early clinical trials have thus far failed to yield an effective reversal agent for these medications, and new treatments are certainly needed. The authors stress that a high index of suspicion for catastrophic taurine inhibitor hemorrhage isaily had a lower rate of intracranial hemorrhage when compared with warfarin, while a higher dose had a similar rate. Additionally, the higherdose dabigatran was superior to warfarin in the annual rate of stroke and systemic embolism. Postulated to by Total hip replacement is classified as at high thrombosis risk. American and French consensus conferences recommended prolonged 35 42 days thromboprophylaxis following THR.
In France, this has been implemented by injection of low glycyrrhetin 471-53-4 molecular weight heparin, with twice weekly platelet monitoring due to the risk of heparin induced thrombopenia. The only oral anticoagulant available was anti vitamin K, compliance could be checked on the International Normalized Ratio. However, difficulty of use, narrow implementation window and risk of hemorrhage, mainly due to misuse, led to AVK being abandoned in most cases as a preventive treatment for postoperative thrombosis in orthopedic surgery. Recently, new oral anticoagulants have come onto the market: dabigatran etexilate, an anti IIa, and rivaroxaban, an anti Xa, with official approval for prescription following THR and total knee replacement. Apixaban, an anti Xa, is to come onto the market soon. They are administered orally and do not require blood tests. In addition to comfort for the patient, who no longer undergoes fludarabine daily injection and weekly blood sampling, cost saving is also significant. These oral anticoagulants raise the issue of compliance for the medical community, as their administration is entirely the responsibility of the patient after discharge home. The present study therefore sought to assess compliance to oral thromboprophylaxis following THR and the possible consequences of non compliance.
The hypothesis was that compliance is good throughout the treatment prescribed for non dependent patients discharged home. Patients and method Patients A prospective continuous cohort study, with university funding and ethics committee approval, included patients undergoing first line THR in the Caen University Hospital Center between November 2, 2009 and July 28, 2010 and stroke receiving oral dabigatran etexilate, at a single dose of two 110 mg capsules per day. Other inclusion criteria were: age 18 75 years, and consent following written and oral information. Exclusion criteria were: discharge to another institution, previous long course anticoagulant treatment, low dabigatran etexilate dose indicated according to the marketing authorization, liver enzyme level more than 2 fold normal threshold, weight less than 50 kg, patient under amiodarone or quinidine, or adult patient under guardianship. Methods Thromboprophylaxis was initiated toward the end of the day of the THR. The study as such began at discharge, for 30 5 days to S30 5.
Disufenton sodium may continue to play a role in treating certain CLL patients due to lower toxicity profiles, convenience as an oral agent, and lower costs. The clinical value of these therapies could be evaluated in additional network meta analyses among different CLL patient populations with varying prognostic factors. Limitations This study has several limitations worth noting. First, a network meta analysis entails extrapolation from available data to indirectly make comparisons that have not actually been conducted. As a result, the validity of indirect estimates generated by a network meta analysis is uncertain. Although taurine requiring additional research, there is evidence to suggest that adjusted indirect comparisons usually agree with results of head to head randomized trials and the validity of the indirect estimates is dependent on the internal validity and homogeneity of the included trials.25 Second, the network meta analysis method is based on model simulation and as with all modeling methods, the results are dependent on data quality and the accuracy of model inputs.
Overall, the studies included in our treatment comparison HSP network received relatively lower Jadad quality scores because they were not double blinded and most did not report the method for randomization. Bias contained in the trials may inflate the estimates of relative and absolute treatment effects. Third, our comparison network was small with only five RCTs and did not assess all available therapeutic options such as bendamustine, pentostatin or cladribine based regimens due to limited published evidence. We conducted a literature search in the Medline database and The Cochrane Library, and also performed manual searches of reference citations. Though this methodology allowed us to conduct a broad search, this review may not have captured all relevant studies. In general, doxorubicin larger comparison networks would allow each study to,borrow more strength, from other studies and could improve the precision and certainty of indirect estimates.
Our comparison network included a three arm trial and our random effects model did not adjust for correlation induced by having more than two arms. Because our network only included one multi arm trial, any potential correlation was likely minimal. If a comparison network contains several multi arm trials, it is recommended that models are developed to account for the potential correlation within each trial.8 Finally, our study was limited to only one endpoint and does not provide information about adverse effects from therapy or therapy administration. Conclusion Among the five therapy options evaluated in this network meta analysis, our results suggest that FCR should be consideredan optimal initial treatment strategy for younger, healthier, treatment naïve CLL patients with low to intermediate risk disease. However, due to limitations of using model simulations, additional evaluations of FCR are necessary in order to clinically validate its therapeutic potential. Conflict of interest David Veenstra has served as a consultant to Genentech, Inc. No other authors declare potential conflicts of interest. Funding Mindy Cheng was supported by a Pharmaceutical Research and Manufacturers of America Foundation pre doctoral fellowship.
Ducreux and colleagues presented preliminary results from a phase II study comparing FOLFIRI with bevacizumab versus dosemodiWed XELIRI and bevacizumab, taurine and demonstrated similar toxicity for the two regimens. In the XELIRI bevacizumab, arm rates of grade 3/4 neutropenia and diarrhea were 17 and 12%, respectively. The results of these two studies are comparable to the rates of grade 3/4 neutropenia and diarrhea noted in our study. These toxicity results compare favorably to the grade 3/4 neutropenia and diarrhea noted in a phase IV study of FOLFIRI and bevacizumab. The eYcacy results noted in our study were also comparable to those noted by Garcia Alfonso and colleagues, and by Ducreux and colleagues. In addition, the median PFS of 11.5 months from our study compares favorably with those from the Hurwitz study, the ECOG 9699 study of FOLFOX or XELOX and bevacizumab, and a phase IV study of FOLFIRI and bevacizumab. In summary, with Disufenton sodium modest dose reduction, the combination of bevacizumab, irinotecan, and capecitabine was well tolerated.
Furthermore, treatment eYcacy was not compromised since outcomes in the current study compared favorably with those from other Wrst line trials. Doxorubicin this combination may be considered another option for Wrst line therapy in patients with mCRC.Microtubules play a fundamental role in diverse cellular functions through a very complex dynamic process of polymerization and depolymerization. Hence, they have emerged as an important target for anticancer drugs. The success of the treatments with taxanes in breast cancer and in other tumor types has led to the development of new microtubule stabilizing agents as antineoplastic drugs. Among them, the epothilones are the furthest along in clinical development. Ixabepilone, the first in a new class of antimicrotubule agents, was approved in the USA and eight other countries both as a monotherapy in metastatic or locally advanced breast cancer after failure of an anthracycline, a taxane, and capecitabine, and as a combination with capecitabine for treatment HSP of metastatic or locally advanced breast cancer resistant to an anthracycline and a taxane.
Peripheral neuropathy, caused by morphologic or functional abnormalities in peripheral nerves, is a nonhematological adverse event associated with all MTSA based chemotherapies, it may be dose limiting and associated with functional impairment. The nature of PN may vary depending on the type of nerve fibers involved: sensory, motor, or autonomic. The incidence of grade 3/4 sensory PN in breast cancer patients treated with taxanes ranges from 0% to 33% and that of grade 3/4 motor neuropathy varies from 0% to 14%. The clinical assessment of neuropathy usually incorporates a careful evaluation of its onset, distribution, severity, and its impact on quality of life. Early diagnosis and management of mild to moderate symptoms are important to prevent progression. Neuropathy is graded by subjective complaints of patients and physical examinations by clinicians. The most widely used grading systems are National Cancer Institute Common Toxicity Criteria, ECOG and WHO criteria. The mechanism of MTSA induced PN is unclear. Preclinical models have demonstrated that both ixabepilone and taxanes produce a very similar neurotoxicity prof.
Therefore, the combination of rapamycin and enzastaurin inhibits relevant targets, induces apoptosis, and reduces cell viability in most SCCHN cell lines. In Vivo Effect of Rapamycin and Enzastaurin on Tumor Growth, Putative Targets, Angiogenesis, Temsirolimus Proliferation, and Apoptosis. We observed the most significant effects on apoptosis and cell viability in the CAL27 cell line in vitro and thus tested whether the combination of rapamycin and enzastaurin was more Gefitinib clinical trial effective than either agent alone in vivo. Rapamycin dose was based on reproducibly inhibiting phospho p70S6K in vivo , and the recommended dose and schedule of enzastaurin on the basis of target inhibition and murine pharmacokinetics 12 was administered.
Both enzastaurin and rapamycin inhibited CAL27 tumor growth compared with vehicle treated control, with the latter more effective as a single agent . Interestingly, the combination had the greatest effect on tumor growth with a greater than 70% relative reduction in mean tumor size. Moreover, toward the end of the experiment tumor growth rate in enzastaurin and vehicle treated mice appeared Gefitinib structure to be equivalent, suggesting that enzastaurin was becoming less effective over time but that this could be overcome by rapamycin coadministration. We determined whether the growth inhibition observed in vivo was associated with inhibition of putative enzastaurin targets. After the mice were killed, tumors were harvested and analyzed for expression of phospho MARCKS and phospho GSK3b . Consistent with the in vitro experiments, enzastaurin inhibited both these markers compared to vehicle control.
As expected, rapamycin had no effect on phospho MARCKS expression and Gefitinib solubility consistent with a feedback activation of Akt, phospho GSK3b increased significantly in tumors from rapamycintreated animals. Both rapamycin and enzastaurin have been associated with inhibition of proliferation and apoptosis therefore, we determined whether the same effects could be observed in vivo. Immunohistochemical staining of tumor tissue from treated animals demonstrated a significant reduction in proliferation in all 3 treatment groups as measured by Ki 67 . However, tumors exposed to enzastaurin and rapamycin did not demonstrate any further decrement in Ki 67 staining compared with single agent treatment implicating an alternative process accounting for the greater efficacy of the combination.
TUNEL staining was used to assess the degree of apoptosis present in each condition. A significant increase in apoptosis was observed in all 3 treatment groups and was especially evident with rapamycin alone or rapamycin and enzastaurin . Both rapamycin and enzastaurin have been noted to possess antiangiogenic activity; the former possibly through inhibition of vascular wealth inequalities endothelial growth factor production13 and the latter likely through a direct inhibition of PKCb, which is a downstream mediator of vascular endothelial growth factor induced endothelial cell proliferation.9 Therefore, we stained sections of harvested tumors for the endothelial cell marker, CD31, and calculated microvessel density . As expected rapamycin treated tumors demonstrated a pronounced reduction in MVD . Surprisingly, tumors from enzastaurin treated mice did not demonstrate a change in MVD.
from the GenBank, accession number AAC83551.1. The sequence and 3D structure of PFV IN was extracted from the Protein Data Bank , and only residues 51– 374 covering the three functional Paclitaxel domains were luded. According to the secondary structure information of the template, the alignment was adjusted manually to obtain a more reasonable matching. And then, based on the sequence alignment result, a 3D model of the HIV 1 IN was generated and refined with the Program Prime from Schrodinger Suite 2008. Construction for the HIV 1 IN–vDNA complex and INSTIs binding model The structure based alignment of PFV IN and HIV 1 IN was used to guide the construction of HIV 1 IN–DNA complexes.
The 19 base pair mimic of pre processed terminal vDNA from the PFV structures and the Mg2þ ions from the inhibited PFV structures were introduced to the HIV 1 IN model by program Swiss Pdb Viewer using the Ca positions of active site residues Asp64, Asp116, and Glu152 from the Procollagen C Proteinase CCD. Similarly, RAL, vDNA, and Mg2þ ions from the PFV structure were fitted into the modeled HIV 1 IN to get the HIV 1 IN–vDNA–RAL complex. Here, it should also be pointed out that vDNA from the PFV structure was mutated to HIV 1 vDNA based on the DNA sequence alignment. Molecular dynamics simulations The MD simulations of the HIV 1 IN–vDNA and HIV 1 IN– vDNA–RAL complexes described above were preformed using the AMBER10.0 program. The strand AMBER force field was used to describe the HIV 1 IN and vDNA. The force field parameter for the inhibitor generated using the Antechamber program in AMBER10.
0 were described by the General objectified Amber Force Field . The partial charges of the inhibitor was determined using the RESP fitting technique based on the optimized structures and the electrostatic potentials calculated using HF/6 31G* in Gaussian09 program. The hydrogen atoms and the missing atoms were added using the Leap module, 29 Na þ counter ions were added to electrically neutralize each systems. Then the two starting structures were immersed in a rectangular prism preequilibrated TIP3P waters with at least 10 A˚ distance around the complex, and about 23351 and 23341 TIP3P waters were added in the HIV 1 IN–vDNA and HIV 1 IN–vDNA–RAL complex, respectively. Prior to MD simulations, two steps of minimization were carried out. In the first stage, we kept the protein fixed with a position restraint of 2.
0 kcal/mol/A˚ 2, and we just minimized the positions of the water molecules. In the second stage, we minimized the entire system by releasing all the restrains. The two minimization stages consisted of 5000 steps in which steepest descent method was used in the first 1000 steps and conjugated gradient method was used in the last 4000 steps. MD trajectories were run using the minimized structure as a starting input. After 50 ps NVT ensemble heating process, the systems were gradually raised from 0 to 300 K using the Langevin dynamics method, and the density of the systems quickly reached 1 g/cm3 during this heating stage. Then, 150 ps equilibrating process was executed at 1 atm and 300 K using the NPT ensemble in three steps of 50 ps. During the three periods of this second stage, the Ca was restrained with harmonic force constant of 2.0, 1.0, and 0 kcal/mol/A˚ 2, respectively.
in either preintegration or postintegration phases of infection . As preintegration latency does not persist during long term ART, we sought to identify postintegration Parietin latency in infected resting CD4 T cells in BLT mice. We first demonstrated the presence of resting CD4 T cells in naive BLT mice. For this analysis, we isolated mononuclear cells from peripheral blood, bone marrow, spleen, lymph nodes, liver, and lung according to published protocols . All mononuclear cells from individual mice were combined and resting human CD4 T cells were negatively selected by magnetic separation essentially using the same approach used to isolate resting cells from human leukapheresis product . Briefly, cells were ubated with antibodies specific for mouse CD45 and TER119 and for human CD8, CD14, CD16, CD19, CD56, glycophorin A, CD41, HLA DR, CD25 , and CD31 and CD105 .
Antibody bound cells were removed using magnetic selection and the flowthrough fraction containing the purified resting cells was collected. This negative selection approach resulted in a 97% pure resting CD4 T cell population as defined by expression of CD4, CCR7, and CD27 and lack of expression Bcl-2 Signaling Pathway of CD8, CD25, HLA DR, and CD11b . After establishing that resting CD4 T cells are present in BLT mice and that they can be isolated with the same procedure used to obtain similar cells from human peripheral blood, we determined whether a latent reservoir of HIV infected cells is present in infected BLT mice on ART. The general approach used to accomplish this objective is described in Fig. 3A.
Administration of ART to infected BLT mice resulted in an average 3.2 log reduction in plasma viral load . Mononuclear cells from peripheral blood, lymph nodes, human thymic organoid, spleen, bone marrow, lungs and liver were collected and all cells from each individual mouse were pooled for resting cell isolation. Resting CD4 T cell enrichment was performed on each pool paraffin of cells, as described above . The average yield of resting cells per BLT mouse was 3.0 106 . To limit the potential contribution of nonintegrated HIV DNA to the outgrowth assay results, the enriched population of resting cells was cultured in the presence of 15 nM efavirenz and 1 Mraltegravir for 2 days prior to any stimulation for viral outgrowth . The lack of ongoing virion production in resting cells maintained in efavirenz and raltegravir was confirmed prior to coculture and stimulation of viral outgrowth as previously published .
The frequency of resting cell infection was determined using a maximum likelihood method, and the results were expressed as infectious units per million resting CD4 T cells . The coculture assay results yielded an average of 9.9 IUPM resting CD4 T cells per mouse . These results demonstrate that HIV infection of humanized mice results in the establishment of a population of resting cells latently infected with HIV that can be readily isolated and induced to express HIV ex vivo, recapitulating key aspects of the human condition. Because of the relatively short frame of time necessary to observe ART efficacy in this model, preclinical evaluation of successful HIV eradication interventions can be rapidly In summary, we have established a novel application of the humanized BLT mouse model for the study.
combinations in HCT116 and HT29 tumour xenografts HCT116 tumour bearing mice were treated as described in the methods section with PXD101 and 5 FU . At these concentrations Tasocitinib each compound alone produced reductions in tumour volume over the measured period . When PXD101 and 5 FU were administered together there was a further inhibition of tumour growth, with a signiWcant reduction in volume at days 28 and 31 compared to the single compound treated groups . At day 24, there was a signiWcant reduction in tumour growth with co administration compared to 5 FU alone only. No signiWcant eVect on body weight was observed for any of the treated groups . In a subsequent xenograft experiment, using HT29 cells, the dose of 5 FU was increased to 30 mg/kg in an attempt to achieve an enhanced eVect over that found using 15 mg/kg 5 FU in the HCT116 study.
Only one cycle, however, of 5 FU treatment was administered at this level due to toxicity. The combination of 100 raltegravir molecular weight mg/ kg PXD101 with 30 mg/kg produced four mice with toxic weight loss, two of which subsequently died. This group was, therefore, excluded from subsequent study. Some tumour inhibition was observed in the 60 mg/ kg PXD101 and 30 mg/kg 5 FU single treatment groups, with increased tumour growth inhibition in the PXD101/5 FU group . The eVect of the combination is larger when data is normalised to the pre treatment tumour volume , with the tumour reduction by the combination signiWcantly greater than the control and 5 FU alone groups at days 24, 28, 31 and 35. Statistical signiWcance was not achieved compared to the PXD101 alone group at any measurement time.
It should be noted that toxic weight chemical screening loss was observed in mice from both the 5 FU Dasatinib ic50 alone and in the group treated with the combination . Owing to this, the 5 FU treatment was halted after only the Wrst weekly cycle allowing body weight recovery, even though they were treated with the second cycle of PXD101. Discussion The Wnding that treatment of cell lines with HDACi in vitro, as demonstrated by others , only appears to regulate a relatively small subset of genes is surprising given the lack of HDAC isoform selectivity that most HDACi demonstrate see ref. for an overview of the classical HDAC family). Commonly included in the subset of genes that are regulated by HDACi is TS.
It has been nausea previously demonstrated that HDACi, including trichostatin A , suberoylanilide hydroxamic acid , MS275, FK228 and BL1521, can down regulate the expression of TS in vitro . Down regulation of TS is likely to be a fundamental response to histone acetylation induced by HDACi treatment since it was not found to be species or cell line/type speciWc .As discussed in the introduction, over expression of TS in colorectal cancer in vitro is implicated in the mechanism of resistance to 5 FU and is a marker of poor prognosis clinically . Therefore any reduction in TS levels in patient colorectal tumours may translate into a clinical advantage. It has been demonstrated here that PXD101, in common with other HDACi, can down regulate TS expression both at the mRNA and protein level. Similar to other HDACi, it is likely that PXD101 regulates TS levels via eVects on histone acetylation and downstream transcription. This led to the hypothesis that combining .
with chemotherapy are under way in various malignancies.25 We recently initiated a phase I/II AZD2171 study of the cyclophosphamide, doxorubicin, and cisplatin regimen plus belinostat in patients with advanced thymic malignancies who have not received prior chemotherapy .The inhibition of histone deacetylase can induce differentiation, growth arrest, and apoptosis in cancer cells. This phase II multicenter study was undertaken to estimate the efficacy of belinostat, a potent inhibitor of both class I and class II HDAC enzymes, for the treatment of myelodysplastic syndrome . Adults with MDS and ≤2 prior therapies were treated with belinostat 1,000 mg/m2 IV on days 15 of a 21 day cycle. The primary endpoint was a proportion of confirmed responses during the first 12 weeks of treatment.
Responding patients could receive additional cycles until disease progression or unacceptable toxicity. Twenty one patients were enrolled, and all were evaluable. Patients were a median 13.4 months from diagnosis, nebivolol molecular weight and 14 patients Doripenem price had less than 5% bone marrow blasts. Seventeen patients were transfusion dependent. Prior therapy included azacytidine and chemotherapy . The patients were treated with a median of four cycles of belinostat. There was one confirmed response hematologic improvement in neutrophils for an overall response rate of 5% . Median overall survival was 17.9 months. Grades 34 toxicities considered at least to be possibly related to belinostat were: neutropenia , thrombocytopenia , anemia , fatigue , febrile neutropenia , headache , and QTc prolongation .
Because the study met the stopping rule in the first stage of enrollment, it was closed to further accrual.MDS are the DNA methyltransferase Gemcitabine Antimetabolites inhibitor inhibitors decitabine and azacytidine, which alter methylation patterns on DNA, leading to changes in gene expression and cellular differentiation. Histone deacetylase inhibitors also exert their activity via epigenetic changes in malignant cells, by promoting acetylation of histones and thereby, altering nucleosome and chromatin structure. Pre clinical studies have shown the HDAC inhibitors induce differentiation in leukemic cell lines and in leukemia cells collected from acute myeloid leukemia patients . Multiple HDAC inhibitors have been studied in MDS and AML, with activity observed in phase I and II studies .
Belinostat is a potent inhibitor of both classes I and II HDAC enzymes, and in vitro studies have demonstrated its ability to inhibit growth and promote apoptosis in cancer cell lines . A phase I study in patients with advanced solid tumors demonstrated hyperacetylation of histone H4 that was sustained for 424 h after each dose of belinostat carbohydrates . That study established a maximum tolerated dose of 1,000 mg/m2 daily for 5 days of a 21 day cycle, with dose limiting toxicities of fatigue, atrial fibrillation, diarrhea, nausea, and vomiting. No significant myelosuppression was observed. A similar toxicity profile was observed in a phase I study of belinostat in patients with advanced hematologic malignancies, with the exception that no cardiac events occurred. There was only one case of grades 34 hematologic toxicity over baseline. This multicenter phase II study was undertaken to estimate the efficacy and the toxicity of belinostat in patients with MDS.
treatment was a rise in PC content, which was time dependent in HT29 cells. When detectable , GPC levels decreased post belinostat treatment. The changes in PC and GPC were confirmed by 1H MRS which indicated that the tCho signal was also significantly higher following Gamma Secretase belinostat treatment in both cell lines. The rise in PC is in line with our previous findings with LAQ824 and SAHA in HT29 cells indicating that it is not inhibitor chemotype specific or cell line/tissue typedependent. Furthermore, belinostat treatment resulted in increased BCAAs, alanine, and threonine levels in both HT29 and PC3 cells as shown by 1H MRS. These changes were also timedependent in HT29 cells.Taken together, these observations suggest that belinostat treatment led to differential metabolic routing that favors the conversion of pyruvate to alanine at the expense of lactate synthesis.
A previous report showed BMS-354825 inhibition of glucose transport concomitant with reduced hexokinase activity following HDAC inhibition . Here, we did not observe a reduction in 13C glucose accumulating in the cells, and the combined level of downstream 13C intermediates formed from 13C glucose seemed to be comparable between control and treated cells . These observations suggest that glucose uptake was probably unaltered under our experimental conditions although more work is required to test this hypothesis. Furthermore, and although neither of the changes in individual content of intracellular 13C lactate or 13C glutamate following belinostat treatment were statistically significant, the glutamate/ lactate ratio increased by approximately 3 fold.
This effect could suggest an altered balance between glycolysis and Krebs cycle metabolism as previously reported . Next, surgery we investigated the basis for the PC rise following belinostat treatment. PC is formed via 2 main routes: de novo synthesis through ChoK catalyzed phosphorylation of its precursor choline and release from membrane PtdCho via PtdCho specific phospholipase C or release of choline from PtdCho via phospholipase D followed by ChoK mediated phosphorylation . To distinguish these 2 processes, we assessed levels of 13CPC in cells incubated in choline for 3 hours. This short duration ensured that the 13C PC observed was formed de novo from exogenous 13C choline, thus excluding contributions from membrane PtdCho derived PC .
13C MRS indicated that the levels of 13C PC formed were approximately 1.5 fold higher in belinostat treated cells relative to controls. The amplitude of this increase is similar to that observed by 1H and 31P MRS indicating that the elevation in steady state PC is driven primarily by increased de novo synthesis. To further delineate the molecular processes driving this effect, we assessed the expression of ChoKa. qRT PCR analysis revealed an induction in ChoKa gene expression in HT29 cells which correlated strongly with the rise in PC levels measured by MRS and which was also confirmed by ChoKa protein expression in these cells. A similar increase in ChoKa expression was also observed in PC3 cells, albeit to a lesser extent. These findings point to the induction of ChoKa expression as a key driver of the rise in PC observed by MRS following belinostat treatment in both HT29 and PC3 cells.