Transport of six tyrosine kinase inhibitors: active or passive ?

Transport of erlotinib, gefitinib, sorafenib, sunitinib, dasatinib and crizotinib can be active or passive, which was studied by measuring uptake at low (4 °C; passive) and normal temperature (37 °C; active and passive) and by the use of specific organic cation transporter (OCT) inhibitors. Intracellular accumulation was determined using Caco-2 as monolayers, while for gut permeation we used differentiated Caco-2 as model for intestinal epithelium in the Transwell system. Sorafenib and crizotinib uptake are likely to be dependent on passive transport. Gefitinib, dasatinib and sunitinib uptake seem to be active. Erlotinib’s transport also seems to be active. This study suggests that hOCTs might be involved in the apical to basolateral transport of gefitinib and crizotinib. Overall it can be concluded that the accumulation and transport of these six TKIs are very different, despite the fact that they are all tyrosine kinase inhibitors

Transport of six tyrosine kinase inhibitors: active or passive ?

Transport of erlotinib, gefitinib, sorafenib, sunitinib, dasatinib and crizotinib can be active or passive, which was studied by measuring uptake at low (4 °C; passive) and normal temperature (37 °C; active and passive) and by the use of specific organic cation transporter (OCT) inhibitors. Intracellular accumulation was determined using Caco-2 as monolayers, while for gut permeation we used differentiated Caco-2 as model for intestinal epithelium in the Transwell system. Sorafenib and crizotinib uptake are likely to be dependent on passive transport. Gefitinib, dasatinib and sunitinib uptake seem to be active. Erlotinib’s transport also seems to be active. This study suggests that hOCTs might be involved in the apical to basolateral transport of gefitinib and crizotinib. Overall it can be concluded that the accumulation and transport of these six TKIs are very different, despite the fact that they are all tyrosine kinase inhibitors

Subcellular localization of several structurally different tyrosine kinase inhibitors

Protein tyrosine kinases form an important target for a new class of anticancer drugs, the tyrosine kinase inhibitors (TKIs). Recently we demonstrated that sunitinib, an inhibitor of the membrane-associated vascular endothelial growth factor receptor (VEGFR), is trapped in lysosomes which isolates the drug from its intended target. Therefore we investigated whether this also holds for other TKIs, targeted against different protein kinases. For this purpose we used the ProteoExtractR kit, which enables a subcellular extraction separating cellular proteins into four distinct fractions covering the cytosol, membranes and membrane organelles (including lysosomes), nuclear proteins and the cytoskeleton. Since TKIs are 98-100 % protein bound we used this property to study their subcellular distribution and used Caco-2 cells as a model. As expected after 2 hours exposure sunitinib was trapped in cytosol (58 %) and organelles (42 % including lysosomes). Crizotinib, an inhibitor of ALK-EML4, showed a similar distribution. However, erlotinib, an inhibitor of the epidermal growth factor receptor (EGFR) showed a very low cellular accumulation and was limited to the organelle fraction. In contrast, the other EGFR inhibitor, gefitinib was predominantly located in the cytosolic (39 %) and membrane fraction (44 %). Sorafenib, another VEGFR inhibitor was predominantly located in the organelle fraction (85 %) and cytosol (15 %) after 2 hours, while after 24 hours distribution decreased (9.9 fold) with a slight shift. Dasatinib, an inhibitor of BCR-Abl was located only in the cytosol (100 %). In general localization after 24 hours was comparable, albeit several small changes were seen. In conclusion protein fractionation with the ProteoExtractR Subcellular Proteome Extraction kit demonstrated large differences in TKI levels in various cellular organelles, with a pattern in agreement with lysosomal accumulation of sunitinib

Cellular pharmacology of multi-and duplex drugs consisting of ethynylcytidine and 5-fluoro-2′-deoxyuridine

In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation

Pemetrexed Induced Thymidylate Synthase Inhibition in Non-Small Cell Lung Cancer Patients: A Pilot Study with 3 '-Deoxy-3 '-[F-18]fluorothymidine Positron Emission Tomography

OBJECTIVES: Pemetrexed is a thymidylate synthase (TS) inhibitor and is effective in non-small cell lung cancer (NSCLC). 3'-deoxy-3'-[¹⁸F]fluorothymidine (¹⁸F-FLT), a proliferation marker, could potentially identify tumor specific TS-inhibition. The aim of this study was to investigate the effect of pemetrexed-induced TS-inhibition on ¹⁸F-FLT uptake 4 hours after pemetrexed administration in metastatic NSCLC patients. METHODS: Fourteen NSCLC patients underwent dynamic ¹⁸F-FLT positron emission tomography (PET) scans at baseline and 4 hours after the first dose of pemetrexed. Volumes of interest were defined with a 41%, 50% and 70% threshold of the maximum pixel. Kinetic analysis and simplified measures were performed. At one, two, four and six hours after pemetrexed, plasma deoxyuridine was measured as systemic indicator of TS-inhibition. Tumor response measured with response evaluation criteria in solid tumors (RECIST), time to progression (TTP) and overall survival (OS) were determined. RESULTS: Eleven patients had evaluable ¹⁸F-FLT PET scans at baseline and 4 hours after pemetrexed. Two patients had increased ¹⁸F-FLT uptake of 35% and 31% after pemetrexed, whereas two other patients had decreased uptake of 31%. In the remaining seven patients ¹⁸F-FLT uptake did not change beyond test-retest borders. In all patients deoxyuridine levels raised after administration of pemetrexed, implicating pemetrexed-induced TS-inhibition. ¹⁸F-FLT uptake in bone marrow was significantly increased 4 hours after pemetrexed administration. Six weeks after the start of treatment 5 patients had partial response, 4 stable disease and 2 progressive disease. Median TTP was 4.2 months (range 3.0-7.4 months); median OS was 13.0 months (range 5.1-30.8 months). Changes in ¹⁸F-FLT uptake were not predictive for tumor response, TTP or OS. CONCLUSIONS: Measuring TS-inhibition in a clinical setting 4 hours after pemetrexed revealed a non-systematic change in ¹⁸F-FLT uptake within the tumor. No significant association with tumor response, TTP or OS was observed

Cellular pharmacology of multi- and duplex drugsconsisting of ethynylcytidine and 5-fluoro-2′-deoxyuridine

Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2′deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK−. ETC-FdUrd was active (IC50 of 2.2 and 79 nM) in FM3A/0 and TK− cells, respectively. ETC-L-FdUrd was less active (IC50: 7 nM in FM3A/0 vs 4500 nM in FM3A/TK−). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC50:19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80–90%), but to a lower extent in FM3A/TK− (10–50%). FdUMP was hardly detected in FM3A/TK− cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation

Experiments for foam model development and validation.

A series of experiments has been performed to allow observation of the foaming process and the collection of temperature, rise rate, and microstructural data. Microfocus video is used in conjunction with particle image velocimetry (PIV) to elucidate the boundary condition at the wall. Rheology, reaction kinetics and density measurements complement the flow visualization. X-ray computed tomography (CT) is used to examine the cured foams to determine density gradients. These data provide input to a continuum level finite element model of the blowing process

Rehabilitation versus surgical reconstruction for non-acute anterior cruciate ligament injury (ACL SNNAP): a pragmatic randomised controlled trial

BackgroundAnterior cruciate ligament (ACL) rupture is a common debilitating injury that can cause instability of the knee. We aimed to investigate the best management strategy between reconstructive surgery and non-surgical treatment for patients with a non-acute ACL injury and persistent symptoms of instability.MethodsWe did a pragmatic, multicentre, superiority, randomised controlled trial in 29 secondary care National Health Service orthopaedic units in the UK. Patients with symptomatic knee problems (instability) consistent with an ACL injury were eligible. We excluded patients with meniscal pathology with characteristics that indicate immediate surgery. Patients were randomly assigned (1:1) by computer to either surgery (reconstruction) or rehabilitation (physiotherapy but with subsequent reconstruction permitted if instability persisted after treatment), stratified by site and baseline Knee Injury and Osteoarthritis Outcome Score—4 domain version (KOOS4). This management design represented normal practice. The primary outcome was KOOS4 at 18 months after randomisation. The principal analyses were intention-to-treat based, with KOOS4 results analysed using linear regression. This trial is registered with ISRCTN, ISRCTN10110685, and ClinicalTrials.gov, NCT02980367.FindingsBetween Feb 1, 2017, and April 12, 2020, we recruited 316 patients. 156 (49%) participants were randomly assigned to the surgical reconstruction group and 160 (51%) to the rehabilitation group. Mean KOOS4 at 18 months was 73·0 (SD 18·3) in the surgical group and 64·6 (21·6) in the rehabilitation group. The adjusted mean difference was 7·9 (95% CI 2·5–13·2; p=0·0053) in favour of surgical management. 65 (41%) of 160 patients allocated to rehabilitation underwent subsequent surgery according to protocol within 18 months. 43 (28%) of 156 patients allocated to surgery did not receive their allocated treatment. We found no differences between groups in the proportion of intervention-related complications.InterpretationSurgical reconstruction as a management strategy for patients with non-acute ACL injury with persistent symptoms of instability was clinically superior and more cost-effective in comparison with rehabilitation management

The Pharmacokinetics of Vitamin A in relation to its Teratogenicity in Healthy Women

The project involved four clinical studies, investigating the various aspects of vitamin A pharmacokinetics following its administration in the three sources most commonly available to the general public i.e. animal liver, vitamin A supplements and transdermal creams. Investigated were the influence of (1) posture and previous dosing, (2) food and dosage, and (3) multiple dosing on the absorption of vitamin A and the formation of its teratogenic metabolites. A fourth study investigated the effect of transdermal vitamin A absorption in healthy women of child-bearing age.Teratogenic concentrations were not found in any of the four studies performed. Posture, previous dosing and multiple dosing did not alter the absorption of vitamin A and the formation of its metabolites. However, the source of Vitamin A ingestion and the effect of dosing in conjunction with food were found to be of significance. Vitamin A and its metabolites levels were found to be many folds higher after vitamin A supplement dosing compared to dosing as a liver meal. Also, vitamin A supplement dosing in conjunction with a meal gave significantly faster absorption compared to dosing on an empty stomach. Long-term, high dose transdermal application of vitamin A creams resulted in negligible systemic amounts of vitamin A and its metabolites. In all four studies, it was observed that there were large inter- and intra-individual variations.Vitamin supplements can be beneficial to the general public when used at the Recommended Daily Allowances. However, most sources of vitamin A are freely available to the general public, either as food sources or as over-the-counter products. Hence, abuse is a possibility, especially in the case of vitamin supplements.</p

Can cytidine deaminase be used as predictive biomarker for gemcitabine toxicity and response?

International audienc