Obligations of Contracts: Intent and Distortion

<p><b>Introduction:</b> Treosulfan is an alkylating agent that is used for the treatment of ovarian cancer and for conditioning prior to stem cell transplantation. It is a prodrug that is activated non-enzymatically to two active epoxides.</p> <p><b>Objectives:</b> To optimize a protocol for both <i>in vivo</i> samples handling and <i>in vitro</i> drug preparation. Treosulfan stability was tested in biological fluids at different conditions as well as for its cytotoxicity on cell lines.</p> <p><b>Results:</b> Plasma samples can be safely frozen for a short period up to 8 h, however; for longer periods, samples should be acidified. Urine samples and cell culture media can be safely frozen regardless their pH. For <i>in vitro</i> investigations, incubation of treosulfan at 37 °C for 24 h activated 100% of the drug. Whole blood acidification should be avoided for the risk of hemolysis. Finally; treosulfan cytotoxicity on HL-60 cells has increased following pre-incubation for 24 h at 37 °C compared to K562 cell line.</p> <p><b>Conclusion:</b> The stability profiling of treosulfan provided a valuable reference for handling of biological samples for both <i>in vivo</i> and <i>in vitro</i> studies. These results can be utilized for further investigations concerning the drug kinetics and dynamics in addition to the development of new pharmaceutical formulations.</p

Effect of fludarabine on <i>POR</i> gene expression.

<p>The gene expression of <i>POR</i> was not significantly changed after treatment with fludarabine.</p

Apparent enzyme kinetic parameters for 4-OH-Cy formation by different microsome batches with recombinant <i>CYP2B6*1</i> and different POR/CYP ratios.

<p>* POR measured as Cytochrome c reductase activity as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141979#pone.0141979.t001" target="_blank">Table 1</a></p><p>** <i>V</i>_<i>max</i> is based on 4-OH-Cy formed</p><p><b><i>CL</i></b>_{<b><i>int</i></b>} Calculated as <b><i>V</i></b>_{<b><i>max</i></b>}<b><i>/K</i></b>_{<b><i>m</i></b>}</p><p>Cyclophosphamide at different concentrations was incubated in 2 independent experiments with 3 batches of CYP2B6.1 containing different POR/CYP ratios. The 4-OH-Cy formation, used as a measure of Cy metabolism, was measured using HPLC. The results shown are the averages obtained for each of the three batches. The results show almost proportional increase of Cy intrinsic clearance (i.e. apparent <i>V</i>_<i>max</i>/<i>K</i>_<i>m</i>, with <i>V</i>_<i>max</i> measured from 4-OH-Cy formation) with higher POR/CYP ratios from Batch 1 to Batch 3.</p

Up-regulation of <i>POR</i> mRNA during Cy conditioning.

<p>The gene expression of <i>POR</i> was significantly (<i>p</i> < 0.001, t-test) up-regulated 24 hours after the first Cy infusion and 6 hours after the second dose of Cy, compared to pre-Cy conditioning. The inter-individual variation in relative POR expression increased from 4.3-fold before Cy conditioning to 9.9-fold before the 2^nd dose of Cy and to 16.5-fold by the end of the treatment. The high variation in up-regulation of <i>POR</i> gene expression during Cy treatment may contribute to the high inter-individual variations in Cy kinetics reported in patients, possibly in part due to different inducibility of the polymorphic forms of <i>POR</i>. Circles in the figure are for four patients with <i>POR*28</i> polymorphism.</p

Characteristics of commercial microsomes containing recombinant human CYP2B6.1 and POR.

<p>Batch 1–3 were microsomes (Bactosomes) from <i>E</i>. <i>Coli</i> containing co-expressed <i>CYP2B6*1</i> and human <i>POR</i>. The POR batch was a control without CYP.</p><p>*Batch 2 also contained a supplement of purified human cytochrome b5</p><p>**P450 concentration (nmol/mL)</p><p>***Protein concentration (mg/mL)</p><p>^#Specific P450 content (pmol/mg protein)</p><p>^##Cytochrome c reductase activity (nmol/min/mg protein) as marker for POR activity</p><p>N/A Not applicable</p><p>Characteristics of commercial microsomes containing recombinant human CYP2B6.1 and POR.</p

Patient characteristics.

<p><b>Abbreviations:</b> ALL: acute lymphoblastic leukemia; AML: acute myeloid leukemia; ATG: antithymocyte globulin; B: B lymphocyte; CLL: chronic lymphoblastic leukemia; CD 34: bone marrow-derived stem cells; CR: complete remission; Cy: cyclophosphamide; Flu: Fludarabine; HSCT: hematopoietic stem cell transplantation; NHL: Non-Hodgkin lymphoma; P: patient; PR: partial remission; T: T lymphocyte; TBI: Total body irradiation;</p><p>^†: survival time. fTBI: fractionated total body irradiation</p><p>Patient characteristics.</p

Michaelis–Menten curves and Hanes-Woolf plots for 4-OH-Cyclophosphamide enzyme kinetics for different batches of CYP2B6.1.

<p>In 2 independent experiments, Cyclophosphamide (at different concentrations) was incubated with 3 batches of <i>Escherichia coli</i> microsomes containing cDNA-expressed CYP2B6.1 (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141979#pone.0141979.t001" target="_blank">Table 1</a>) with different POR/CYP ratios. The CYP concentration per mg protein and the protein concentration per mL incubation were similar between batches. The 4-OH-Cy formation was measured using HPLC with fluorescence detector. The results shown are the averages obtained with each of the three batches. Fitting the data to Michaelis-Menten kinetics gave an apparent intrinsic clearance, <i>CL</i>_<i>int</i> (<i>V</i>_max/<i>K</i>_m), of 3.1, 25.1, 33.7 μL/min/nmol CYP for Batch 1, Batch 2 and Batch 3, respectively. The intrinsic clearance thus increased with increasing POR/CYP ratio. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141979#pone.0141979.g002" target="_blank">Fig 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141979#pone.0141979.t003" target="_blank">Table 3</a>.</p

Gene clusters in relation to Cy treatment.

<p>The expression of Cy treatment specific genes at 6(30 h) was normalized to the pre-treatment and divided to the following clusters: Cluster 1 showed highly down-regulated genes throughout the treatment (A). Cluster 2 showed highly up-regulated genes throughout the treatment (B). Cluster 3 showed early up-regulated but later normalized genes (C). Cluster 4 showed moderately up-regulated genes (D).</p

Pathways reported in each cluster and genes involved in each of them.

<p>Pathways reported in each cluster and genes involved in each of them.</p

Effect of cyclophosphamide treatment on ANGPTL1 and c-JUN.

<p>The relative expression as measured by qRTPCR (normalized to GAPDH) of disease-related up-regulated genes, ANGPTL1 (A) and c-JUN (B), compared to normal subjects. Cy treatment did not affect the up-regulation of ANGPTL1 and c-JUN.</p