Significance and clinical impact of routinely tested urinary ethyl glucuronide after liver transplantation – development of a risk score

Alcohol abuse after liver transplantation can seriously impact graft and patient survival. However, to date, there is no defined standard procedure to identify patients consuming alcohol after liver transplantation. The aim of this study was to analyze the diagnostic value and clinical impact of routinely measured urinary ethyl glucuronide (uEtG) – a metabolite of ethanol – in patients after liver transplantation. Data of 362 consecutive patients after liver transplantation who visited the University Hospital of Tuebingen for outpatient follow-up were analyzed. Forty-eight patients (13%) displayed positive uEtG results. The uEtG positive group contained significantly more patients with pretransplant alcoholic liver disease. However, two thirds of the uEtG positive patients had no history of pretransplant alcoholic liver disease. Several clinical parameters were significantly associated with positive uEtG. In order to enable a more cost-effective application of uEtG in the future, a clinical risk score was developed (specificity 0.95). In conclusion, routine testing for uEtG reveals a considerable percentage of patients practicing alcohol intake after liver transplantation. Application of our proposed risk score could help focusing uEtG testing on patients at risk.     

Chemotherapy-Associated Liver Injuries: Unmet Needs and New Insights for Surgical Oncologists

Annals of Surgical Oncology -     

Adhesiveness of human uterine epithelial RL95-2 cells to trophoblast: rho protein regulation

Embryo implantation involves adhesion of trophoblast cells to the epithelial lining of the endometrium. Using an in-vitro model to simulate this initial interaction, we previously reported that attachment of human trophoblast-like JAR spheroids to human uterine epithelial RL95-2 cells provokes a Ca(2+) influx in RL95-2 cells depending on apically localized integrin receptors. Here, we demonstrate that adhesiveness of RL95-2 cells for JAR spheroids, measured by a centrifugal force-based adhesion assay, is dependent on Rho GTPases, most likely RhoA. Cellular expression and distribution of RhoA were studied by fluorescence confocal microscopy, focusing on the localization of RhoA and F-actin within the adhesion sites between JAR and RL95-2 cells. Contact areas contained high amounts of RhoA and F-actin fibres near the plasma membrane. To determine whether Rho GTPases may influence JAR cell binding, we treated RL95-2 cells with Clostridium difficile toxin A, which specifically inactivates Rho GTPases. Toxin A treatment changed the subcellular distribution of endogenous RhoA in RL95-2 cells and altered RhoA and F-actin colocalization. Adhesion of JAR spheroids to RL95-2 cells treated with toxin A was largely suppressed. These data indicate that Rho GTPases, most likely RhoA, play an important role in uterine epithelial RL95-2 cells for trophoblast binding, and suggest that RhoA may be involved in local signalling cascades during early embryo implantation in vivo.     

Analysis of receptor-G protein interactions in permeabilized cells

Receptor-induced binding of the stable GTP analogue, guanosine 5-[-thio]triphosphate (GTP [S]), to guanine nucleotide-binding regulatory proteins (G proteins) was measured in various permeabilized cells. In myeloid differentiated human leukemia (HL-60) cells, permeabilized with either digitonin, streptolysin O or Staphylococcus aureus -toxin, binding of GTP [S] induced by three distinct chemoattractant receptors was observed. The extent of receptor-stimulated GTP [S] binding (maximally about 2-fold) was independent of the type of permeabilizing agent used. In human erythroleukemia cells permeabilized with digitonin, agonist activation of thrombin and neuropeptide Y receptors increased GTP [S] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with digitonin, activation of the stably expressed human muscarinic m3 receptor increased GTP[S] binding by about 1.6-fold. In digitonin-permeabilized HL-60 cells, a quantitative analysis of formyl peptide receptors and interacting G proteins was performed. About 50,000 formyl peptide receptors per cell were detected. Agonist binding to these receptors was fully sensitive to regulation by guanine nucleotides and pertussis toxin. The number of high-affinity GTP [S] binding sites, most likely representing heterotrimeric G proteins, was calculated to be about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP [S] binding sites, indicating a ratio of about three activated G proteins per one agonist-activated receptor.Overall, this study indicates that receptor-stimulated GTP [S] binding to G proteins in permeabilized cells is a sensitive and rapid method for analyzing receptor-G protein interactions, which can be applied to a variety of cultured cells and for various receptor systems.     

Hydroxypropyl-β-Cyclodextrin Increases the Aqueous Solubility and Stability of Pilocarpine Prodrugs

Purpose. The effects of 2-hydroxypropyl--cyclodextrin (HP--CD) on the aqueous solubility and stability of two lipophilic bispilocarpine prodrugs were investigated at pH 7.4. Methods. The solubility of prodrugs was studied by phase-solubility method (0–72.5 mM HP--CD). The stability of one of the prodrugs was investigated as a function of temperature (40°C–70°C) and HP--CD concentration (0–72.5 mM). The apparent rate constants (k ₁, k ₂) for degradation of prodrug in 1:1 and 1:2 inclusion complexes and apparent stability constants (K _1:1, K _l:2) were calculated by the curve-fitting method. Results. The phase-solubility diagrams were classified as A_p-type and the apparent stability constants (K _l:l, K _l:2) for 1:1- and 1:2-inclusion complexes were calculated to be 143–815 M^–l and 29–825 M^–1, respectively. The stability of prodrug increased as a function of HP--CD concentration over the studied temperature range. The shelf-life (t _90%, calculated by the Arrhenius equation) of the prodrug in 72.5 mM HP--CD solution increased 5.1-fold and 6.1-fold at 25°C and 4°C, respectively. Conclusions. The solubility of the prodrugs was shown to increase markedly in phase-solubility studies. The degradation rate of prodrug in stability studies was shown to be slower in the l:2-complex than in the l:l-complex and the relative amounts of complex species were found to be dependent on CD concentration.     

Pharmacokinetics of 4′-O-tetrahydropyranyladriamycin given on a weekly schedule in patients with advanced breast cancer

Improved quality of life has gained importance over shortly lasting remissions in yet incurable metastatic breast cancer. Fractionation of drug administration is one of the possible approaches to reduce the concentration-dependent toxicity of anthracyclines. We evaluated the pharmacokinetics of 4-O-tetrahydropyranyladriamycin (THP-ADM) under weekly administration in patients with advanced breast cancer (dose escalation, from 20 to 27 mg/m₂ THP-ADM). The concentration-time curves of THP-ADM in plasma were best described by an open three-compartment model [half-life of the first disposition phase (t_1/2), 3.15 min; terminal elimination half-life (t _1/2), 13.9 h] with a mean area under the curve (AUC) of 12.2 ng h ml^–1mg^–1m^–2, resulting in a mean plasma clearance of 86.91 h^–1m^–2. Metabolism included the formation of Adriamycin (ADM), Adriamycinol (ADM-OH), 13-dihydro-4-O-tetrahydropyranyladriamycin (THP-OH), 7-deoxyadriamycinone (7H-ADn), and 7-deoxy-13-dihydroadrimycinone (7H-ADn-OH), with maximal plasma concentrations ranging from 2.8 to 5.5 ng/ml. The mean total amount of cytotoxic anthracyclines excreted into urine, mainly as the parent drug, was 5% of the delivered dose. ADM and ADM-OH, but not the parent drug, were observed in urine at up to 4 weeks after the last therapeutic cycle. There was a significant correlation between the leukocyte nadir under therapy and the AUC of ADM-OH (r=0.800,P<0.05). Since no shift in the plasma kinetics was observed from the first to the sixth cycle, the favorable ratio of the AUCs of THP-ADM and ADM after fractionation of THP-ADM suggests lower toxic side effects attributable to ADM. This hypothesis was confirmed in a clinical study, where no severe cardiotoxicity and only mild alopecia were observed in 19 patients. Thus, pharmacokinetics studies might be helpful in both individualization of therapy with THP-ADM and optimization of the administration schedule.     

Chronic motor neuropathies: response to interferon-beta1a after failure of conventional therapies

OBJECTIVES: The effect of interferon-beta1a (INF-beta1a; Rebif) was studied in patients with chronic motor neuropathies not improving after conventional treatments such as immunoglobulins, steroids, cyclophosphamide or plasma exchange. METHODS: A prospective open study was performed with a duration of 6-12 months. Three patients with a multifocal motor neuropathy and one patient with a pure motor form of chronic inflammatory demyelinating polyneuropathy were enrolled. Three patients had anti-GM1 antibodies. Treatment consisted of subcutaneous injections of IBF-beta1a (6 MIU), three times a week. Primary outcome was assessed at the level of disability using the nine hole peg test, the 10 metres walking test, and the modified Rankin scale. Secondary outcome was measured at the impairment level using a slightly modified MRC sumscore. RESULTS: All patients showed a significant improvement on the modified MRC sumscore. The time required to walk 10 metres and to fulfil the nine hole peg test was also significantly reduced in the first 3 months in most patients. However, the translation of these results to functional improvement on the modified Rankin was only seen in two patients. There were no severe adverse events. Motor conduction blocks were partially restored in one patient only. Anti-GM1 antibody titres did not change. CONCLUSION: These findings indicate that severely affected patients with chronic motor neuropathies not responding to conventional therapies may improve when treated with INF-beta1a. From this study it is suggested that INF-beta1a should be administered in patients with chronic motor neuropathies for a period of up to 3 months before deciding to cease treatment. A controlled trial is necessary to confirm these findings.