Selective but not pan-CDK inhibition abrogates 5-FU-driven tissue factor upregulation in colon cancer.

Thromboembolic events are complications in cancer patients and hypercoagulability has been linked to the tissue factor (TF) pathway, making this an attractive target. Here, we investigated the effects of chemotherapeutics and CDK inhibitors (CDKI) abemaciclib/palbociclib (CDK4/6), THZ-1 (CDK7/12/13), and dinaciclib (CDK1/2/5/9) alone and in combination regimens on TF abundance and coagulation. The human colorectal cancer (CRC) cell line HROC173 was treated with 5-FU or gemcitabine to stimulate TF expression. TF+ cells were sorted, recultured, and re-analyzed. The effect of treatment alone or in combination was assessed by functional assays. Low-dose chemotherapy induced a hypercoagulable state and significantly upregulated TF, even after reculture without treatment. Cells exhibited characteristics of epithelial-mesenchymal transition, including high expression of vimentin and mucin. Dinaciclib and THZ-1 also upregulated TF, while abemaciclib and palbociclib downregulated it. Similar results were observed in coagulation assays. The same anticoagulant activity of abemaciclib was seen after incubation with peripheral immune cells from healthy donors and CRC patients. Abemaciclib reversed 5-FU-induced TF upregulation and prolonged clotting times in second-line treatment. Effects were independent of cytotoxicity, senescence, and p27kip1 induction. TF-antibody blocking experiments confirmed the importance of TF in plasma coagulation, with Factor XII playing a minor role. Short-term abemaciclib counteracts 5-FU-induced hypercoagulation and eventually even prevents thromboembolic events.

Is Moxifloxacin a Treatment Option for Pancreatic Infections? A Pharmacometric Analysis of Serum and Pancreatic Juice.

Postoperative local infection is a major complication after pancreatic surgery. The aim of this prospective clinical trial was to assess the potential of moxifloxacin (MXF) to treat pancreatic infections from a pharmacokinetic (PK)/pharmacodynamic (PD) perspective. The PK of MXF in serum and pancreatic juice, via an inserted tube in the pancreatic duct, was determined in 19 patients up to day 7 after pancreatoduodenectomy. PK data in both specimens was analyzed with NONMEM 7.3. Intraoperative swipes were performed for microbiological examination. PK/PD target attainment was assessed in both matrices using unbound area under the plasma concentration-time curve/minimum inhibitory concentration (MIC) targets of ≥30 and ≥100, for gram-positive and gram-negative pathogens, respectively. A 2-compartment population PK model in which the measurements in pancreatic juice were assigned to a scaled peripheral compartment best described the PK in both specimens simultaneously. Median (10th-90th percentile) area under the plasma concentration-time curve values after the third dose were 28.9 mg · h/L (18.6-42.0) in serum and 55.8 mg · h/L (23.7-81.4) in pancreatic juice. Target attainment rate for the intraoperatively isolated bacterial strains was ≥0.88 after the third MXF dose. For gram-negatives, high probability of target attainment ≥0.84 was observed in serum for MIC ≤ 0.125 mg/L and in pancreatic juice for MIC ≤ 0.25 mg/L. For gram-positives, the probability of target attainment was 0.84-1 in serum for MIC ≤ 0.5 mg/L and in pancreatic juice for MIC ≤ 1 mg/L. In conclusion, penetration of MXF into pancreatic juice was substantial. The PK/PD analysis indicated that treatment of pancreatic infections by isolates with MIC ≤ 0.25 mg/L (gram-negative) and ≤1 mg/L (gram-positive) should be evaluated in further studies.

Adipose-derived mesenchymal stem cells release microvesicles with procoagulant activity.

Extracellular vesicles are produced by a number of different cell types, among them mesenchymal stromal/stem cells (MSC) of different sources. It has been shown that extracellular vesicles of MSC exert similar therapeutic effects as the cells themselves. Here, we isolated and characterized extracellular vesicles produced by adipose-derived MSC (adMSC) in vitro upon stimulation with the proinflammatory substances lipopolysaccharide (LPS) and tumor necrosis factor (TNF). We found that the number of vesicles produced by adMSC does not change upon stimulation of the cells with LPS and TNF. Furthermore, adMSC-derived extracellular vesicles exert procoagulant activity independent of previous stimulation with LPS or TNF. We found evidence that the vesicles induce coagulation via both the intrinsic and the extrinsic pathway of coagulation.

New coated SPME fibers for extraction and fast HPLC determination of selected drugs in human blood.

Polythiophene (PTh) and polypyrrole (PPy) as sorbent phases for solid phase microextraction (SPME) were applied in order to extract the multi-resistant Staphylococcus aureus (MRSA) antibiotic drugs (linezolid and daptomycin) from whole blood followed by high performance liquid chromatography (HPLC) determination with UV detection. Relative standard deviations (RSDs) of in vitro and pseudo in vivo measurements performed in whole blood were in the range of 4.58-15.91% and 6.09-17.33% for linezolid and daptomycin, respectively. Determination coefficients (R(2)) were in range of 0.9884-0.9945 and 0.9807-0.9818 for linezolid and daptomycin, respectively. This study proved better adsorption capacity of PTh SPME coating compared to PPy coating for both, linezolid and daptomycin.

Polypyrrole solid phase microextraction: A new approach to rapid sample preparation for the monitoring of antibiotic drugs.

Simple or even rapid bioanalytical methods are rare, since they generally involve complicated, time-consuming sample preparation from the biological matrices like LLE or SPE. SPME provides a promising approach to overcome these limitations. The full potential of this innovative technique for medical diagnostics, pharmacotherapy or biochemistry has not been tapped yet. In-house manufactured SPME probes with polypyrrole (PPy) coating were evaluated using three antibiotics of high clinical relevance - linezolid, daptomycin, and moxifloxacin - from PBS, plasma, and whole blood. The PPy coating was characterised by scanning electron microscopy. Influences of pH, inorganic salt, and blood anticoagulants were studied for optimum performance. Extraction yields were determined from stagnant media as well as re-circulating human blood using the heart-and-lung machine model system. The PPy-SPME fibres showed high extraction yields, particularly regarding linezolid. The reproducibility of the method was optimised to achieve RSDs of 9% or 17% and 7% for SPME from stagnant or re-circulating blood using fresh and re-used fibres, respectively. The PPy-SPME approach was demonstrated to meet the requirements of therapeutic monitoring of the drugs tested, even from re-circulating blood at physiological flow rates. SPME represents a rapid and simple dual-step procedure with potency to significantly reduce the effort and expenditure of complicated sample preparations in biomedical analysis.

Determination of antibiotic drug concentrations in circulating human blood by means of solid phase micro-extraction.

BACKGROUND: Promising results in animals have shown the diagnostic potential of polypyrrole coated SPME fibres introduced directly into the blood stream. This study was intended to extend this technique to a clinically relevant antibiotic drug under close to physiological conditions in human blood. METHODS: An artificial vein system was built up from heart and lung machine components. Determination of Linezolid (0-15 mug/mL) was performed by SPME from the flowing system ("online", flow velocities 2-50 cm/s), from blood withdrawn from the system ("offline") and by means of a SPE/HPLC method. SPME was done using new fibres ("new") for each analysis, and in the way that one fibre was reused ("re") for one series of measurements. RESULTS: Drug SPME did not depend on blood flow velocities. Linear regression of data (concentration vs. amount extracted) yielded R(2)=0.998 for SPE/HPLC, R(2)=0.955 for SPME(online_new), 0.929 for SPME(online_re), 0.929 SPME(offline_new), 0.973 for SPME(offline_re), RSD were 52% (SPME(online_new)), 10% (SPME(online_re)), 47% (SPME(offline_new)), 18% (SPME(offline_re)), 8% (SPE/HPLC). CONCLUSIONS: In-vein SPME has the potential to minimize blood requirement for diagnostic purposes and to speed up analysis of clinically relevant drugs, if inter-fibre variation can be reduced through standardized manufacturing.

Penetration of moxifloxacin into the human pancreas following a single intravenous or oral dose.

OBJECTIVES: Failure to prevent secondary infectious complications in acute necrotizing pancreatitis (ANP) is attributable in part to the limited penetration of antimicrobial drugs. As newer quinolones are particularly attractive owing to their antimicrobial activity, for the first time we studied the penetration of moxifloxacin into pancreatic tissue in patients. PATIENTS AND METHODS: In this prospective, non-comparative clinical trial, 60 patients undergoing elective pancreas resection received a single oral or intravenous (iv) dose of 400 mg moxifloxacin for perioperative antimicrobial prophylaxis. The concentration of moxifloxacin was measured in samples taken from blood and from pancreatic tissue at the beginning and at the end of resection. RESULTS: Mean moxifloxacin concentrations in pancreatic tissue following iv or oral administration were 3.1+/-0.9 and 2.7+/-1.4 mg/kg at 3-3.7 h post-dose (first sampling) and 3.6+/-1.5 and 3.1+/-1.8 mg/kg at 4.3-5.3 h post-dose (second sampling), respectively. Corresponding mean plasma concentrations of moxifloxacin were 1.8+/-0.5 and 1.2+/-0.6 mg/L (first sampling) and 1.5+/-0.4 and 1.0+/-0.5 mg/L (second sampling), respectively. From first to second sampling, the mean tissue-to-plasma ratios varied from 1.8+/-0.6 to 2.6+/-1.2 (iv) and from 2.4+/-0.8 to 3.1+/-1.2 (oral). Pancreatic tissue concentrations of moxifloxacin exceeded the MIC90 for the relevant pathogens covered by moxifloxacin for at least 5 h after dosing. CONCLUSIONS: Moxifloxacin has been demonstrated to penetrate efficiently into human pancreatic tissue following iv or oral administration. From a pharmacological perspective, moxifloxacin appears to be promising for prophylaxis and treatment of local pancreas infections. Whether it is beneficial in the prevention and therapy of infectious complications in patients with ANP should be investigated in a controlled clinical trial.

Penetration of ertapenem into different pulmonary compartments of patients undergoing lung surgery.

Ertapenem is approved for the treatment of community-acquired pneumonia (CAP), but its in vivo penetration into lung tissue (LT), epithelial lining fluid (ELF), and alveolar cells (AC) is unknown. Fifteen patients undergoing thoracotomy were treated with 1 g intravenously for perioperative prophylaxis. Bronchoalveolar lavage was performed 1, 3, and 5 hours after ertapenem infusion. Normal LT was sampled at the time of lung extraction. Blood was collected before and at different time points up to 24 hours after infusion. Mean concentrations of ertapenem in plasma, ELF, and AC were at 1.0 hour, 63.1, 4.06, 0.004 mg/L; at 3.0 hours, 39.7, 2.59, 0.003 mg/L; and at 5.0 hours, 27.2, 2.83, 0.007 mg/L. Mean (range) concentration in LT was 7.60 (2.5-19.4) mg/kg tissue 1.5 to 4.5 hours after infusion. In plasma, ertapenem exhibited a Cmax of 94.7 +/- 23.3 mg/L and an AUC(0-last) of 501.1 +/- 266.3 mg x h/L. These results, combined with the reported (MIC)90 of most CAP bacteria, support the previously observed clinical efficacy of ertapenem in the treatment of CAP.