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Introduction: At the time of renal replacement therapy, approximately 20% of critically ill patients have septic shock. In this study, medium cutoff (MCO) continuous venovenous hemodialysis (CVVHD) was compared to high-flux membrane continuous venovenous hemodiafiltration (CVVHDF) in terms of hemodynamic improvement, efficiency, middle molecule removal, and inflammatory system activation. Methods: This is a monocenter crossover randomized study. Between December 31, 2017, and December 31, 2019, 20 patients with septic shock and stage 3 acute kidney injury (AKI) admitted to 2 Italian ICUs were enrolled. All patients underwent CVVHD with Ultraflux® EMiC®2 and CVVHDF with AV1000S® without washout. Each treatment lasted 24 h. Results: Compared to AV1000S®-CVVHDF, EMIC®2-CVVHD normalized cardiac index (β = −0.64; p = 0.02) and heart rate (β = 5.72; p = 0.01). Interleukin-8 and myeloperoxidase removal were greater with AV1000S®-CVVHDF than with EMiC®2-CVVHD (β = 0.35; p < 0.001; β = 0.43; p = 0.03, respectively). Leukocytosis improved over 24 h in EMiC®2-CVVHD-treated patients (β = 4.13; p = 0.03), whereas procalcitonin levels decreased regardless of the modality (β = 0.89; p = 0.01) over a 48-h treatment period. Reduction rates, instantaneous plasmatic clearance of urea, creatinine, and β2-microglobulin were similar across modalities. β2-Microglobulin removal efficacy was greater in the EMiC®2 group (β = 0–2.88; p = 0.002), while albumin levels did not differ. Albumin was undetectable in the effluent in both treatments. Discussion: In patients with septic shock and severe AKI, the efficacy of uremic toxin removal was comparable between MCO-CVVHD and CVVHDF. Further, MCO-CVVHD was associated with improved hemodynamics. Fraction of filtration and transmembrane pressure reduction and the maintenance of equal efficacy might be the key features of CVVHD with MCO membranes in critically ill patients.

Introduction: Few data are available regarding intraoperative plasma concentrations of vancomycin administered as prophylaxis in pediatric cardiac surgery. The aims of this study were to investigate during pediatric cardiac surgery with cardiopulmonary bypass(CPB) the attainment of the area-under-the-curve of the vancomycin serum concentrations versus time over surgery to minimum inhibitory concentration ratio(AUCintra/MIC) of 400 (mg × h)/l and/or a target concentration of 15–20 mg/l. Methods: In a prospective study, 40 patients divided into four subgroups (neonates, infants, children Results: The median (interquartile range) age was 241.5 days (47–3898) and the median weight was 7.1 kg (3.1–37). The median AUCintra/MIC was 254.73 (165.89–508.06). In 11 patients the AUCintra/MIC target was not reached. Neonates displayed the lowest AUCintra/MIC values, and these were significantly lower than those of children ⩾10 years old (p = 0.02). Vancomycin concentrations were above the maximal target of 20 mg/l in 82.5% and 80% of patients at surgery and CPB start, respectively. At CPB and surgery end, 42.5% of patients showed vancomycin concentrations above 20 mg/l and 42.5% below 15 mg/l. Patients⩾10 years old showed the highest peak values whereas neonates were those with the lowest troughs. AUCintra/MIC correlated with age(r:0.36, p = 0.02), weight(r:0.35, p = 0.03), intraoperative protein value(r:0.40, p = 0.01), CPB priming volume/kg(r:−0.33, p = 0.04), CPB duration(r:0.36, p = 0.02) and vancomycin troughs(r:0.35, p = 0.04). Conclusions: An AUCintra/MIC ⩾400 target was not reached in one-quarter of children undergoing heart surgery. Vancomycin peaked before the start of surgery and neonates were those with the lowest troughs. Vancomycin concentrations are affected by CPB hemodilution and by patients’ age and weight.

Pentamidine (PTM), which is a diamine that is widely known for its antimicrobial activity, is a very interesting drug whose mechanism of action is not fully understood. In recent years, PTM has been proposed as a novel potential drug candidate for the treatment of mental illnesses, myotonic dystrophy, diabetes, and tumors. Nevertheless, the systemic administration of PTM causes severe side effects, especially nephrotoxicity. In order to efficiently deliver PTM and reduce its side effects, several nanosystems that take advantage of the chemical characteristics of PTM, such as the presence of two positively charged amidine groups at physiological pH, have been proposed as useful delivery tools. Polymeric, lipidic, inorganic, and other types of nanocarriers have been reported in the literature for PTM delivery, and they are all in different development phases. The available approaches for the design of PTM nanoparticulate delivery systems are reported in this review, with a particular emphasis on formulation strategies and in vitro/in vivo applications. Furthermore, a critical view of the future developments of nanomedicine for PTM applications, based on recent repurposing studies, is provided. Graphical abstract Created with BioRender.com

Cutaneous melanoma is one of the most aggressive solid tumors, with a low survival for the metastatic stage. Currently, clinical melanoma treatments include surgery, chemotherapy, targeted therapy, immunotherapy and radiotherapy. Of note, innovative therapeutic regimens concern the administration of multitarget drugs in tandem, in order to improve therapeutic efficacy. However, also, if this drug combination is clinically relevant, the patient’s response is not yet optimal. In this scenario, nanotechnology-based delivery systems can play a crucial role in the clinical treatment of advanced melanoma. In fact, their nano-features enable targeted drug delivery at a cellular level by overcoming biological barriers. Various nanomedicines have been proposed for the treatment of cutaneous melanoma, and a relevant number of them are undergoing clinical trials. In Italy, researchers are focusing on the pharmaceutical development of nanoformulations for malignant melanoma therapy. The present review reports an overview of the main melanoma-addressed nanomedicines currently under study in Italy, alongside the state of the art of melanoma therapy. Moreover, the latest Italian advances concerning the pre-clinical evaluation of nanomedicines for melanoma are described.

Objective To evaluate the efficacy of a reinforcement message (RM) administered by a hospital pharmacist on adherence, through a randomised study involving patients undergoing oral chemotherapy from which an objective outcome measure and patients' subjective opinions were collected. A secondary aim was to detect which psychological or clinical factors influence adherence. Methods Forty patients were enrolled and randomised to an experimental group (EG) or a control group (CG). The EG received a 10-minute RM provided by a hospital pharmacist with a doctor and a nurse. The CG received the standard of care. To measure adherence, plasma drug concentration and subjective evaluation were taken during the visits, in addition to a psychological assessment (coping strategies, psychological distress and personality traits). Results The EG reported higher drug levels and a statistically significant higher mean score on the subjective evaluation. A linear regression model highlighted statistically significant differences in the plasma drug concentration, after considering toxicity and dose reduction and controlling for the Reward Dependence Scale of the Temperament and Character Inventory between the EG and the CG. Conclusion Adequate information and education on the therapy, using an RM strategy provided by a hospital pharmacist, seems to positively influence adherence to the treatment.

Hyaluronic acid (HA) is a ubiquitous biopolymer involved in many pathophysiological roles. One HA receptor, the cluster of differentiation CD44 protein, is often overexpressed in tumor cells. As such, HA has attracted considerable interest in the development of drug delivery formulations, given its intrinsic targetability toward CD44 overexpressing cells. The present study is focused on examining the correlation of HA molecular weight with its targetability properties. A library of conjugates obtained by linking the amino group of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) to the carboxylic residues of HA of different molecular weight (6.4, 17, 51, 200, and 1,500 kDa) were synthesized and fully characterized. The HA-DMPE conjugates were then used to non-covalently functionalize the highly hydrophobic single-walled carbon nanotubes (CNT), and further encapsulate the anticancer drug doxorubicin (DOX). Our results show that the complexes DOX/CNT/HA-DMPE maintain very good and stable dispersibility. Drug release studies indicated a pH-responsive release of the drug from the nanocarrier. Cell viability tests demonstrated that all HA modified CNTs have good biocompatibility, and specific targeting toward cells overexpressing the CD44 receptor. Among all the molecular weights tested, the 200 kDa HA showed the highest increase in cellular uptake and cytotoxic activity. All these promising attributes make CNT/HA200-DMPE a “smart” platform for tumor-targeted delivery of anticancer agents.

Background and aims The oral administration of insulin has so far been precluded by gastro-intestinal enzyme degradation and poor intestinal absorption. Preliminary evidence for peptide uptake by the gut has recently been obtained, by our research group, following the administration of nanostructured lipid-carrier suspensions loaded with glargine insulin in healthy animal models. Methods and results In this experimental study, glargine insulin-loaded nanostructured lipid carriers have been converted into solid oral dosage forms (tablets, capsules), that are more suitable for administration in humans and have prolonged shelf-life. The liquid and solid oral dosage forms were tested for glargine insulin uptake and glucose responsivity in healthy and streptozotocin-induced diabetic rats (6 animals in each group). A suitable composition gave redispersible solid oral dosage forms from glargine insulin-loaded carriers, using both spray-drying and freeze-drying. It was observed that the liquid and solid formulations had relevant hypoglycaemic effects in healthy rats, while only capsules were efficacious in diabetic rats; probably because of gut alterations in these animal models. Detected glargine insulinaemia was consistent with a glycaemic profile. Conclusion The formulations under study showed their potential as oral glucose-lowering agents, particularly when used as capsules. However, further study is needed to produce a useful orally-active insulin preparation.

The management of infection in the intensive care unit represents an imperative challenge for critical care clinicians. At present, antibiotic dosing regimens are derived from studies on healthy volunteers and do not account for these major differences in drug prescriptions. The chapter summarizes the pharmacokinetic/pharmacodynamics relationship changes in antimicrobial agents resulting from the typical homeostatic disturbance or altered end-organ function of the critical illness. The focus is on how the renal clearance alterations or continuous renal replacement therapy may affect individual antimicrobial dosage and dosing interval of the antimicrobial agents.

Some approaches to enhance the entrapment efficiency within nanoparticulate systems, chiefly of hydrophilic molecules, developed mainly by the Turin University Pharmaceutical Technology group, are discussed. Several approaches have been developed: the first entails associating anticancer hydrophilic molecules to liposomes, developing lipophilic prodrugs of these molecules, and encapsulating them in liposomes. The transformation of hydrophilic drugs into lipophilic prodrugs can also overcome problems of poor entrapment efficiency and rapid release from polymer nanocarriers. Examples are nanospheres and nanocapsules produced from various PEGylated poly(alkylcyanoacrylate) copolymers. Strategies have also been developed to enhance hydrophilic drug entrapment in solid lipid nanoparticles (SLN). Hydrophobic ion pairing was designed to enable various antitumor drugs to be entrapped in SLN, produced by the coacervation method. Another technique comprises the covalent linkage of antitumoral or antiviral drugs to a squalenoyl-derived chain, affording bioconjugates that self-assemble as stable nanoparticles. A further development comprises mesoporous silica nanoparticles with immobilized hydrophilic antioxidants, for topical applications: they were complexed with hydrophilic antioxidants (Trolox ® or rutin). Polymer-shelled and perfluoropentane-cored nanobubbles have also been designed, as versatile multifunctional carriers for the delivery of gases, drugs, and genes; the size range is below 500 nm, with shell thickness in the 30–50 nm range.

Background Herbal medicines (HM) are those with active ingredients made from plant parts, such as leaves, roots or flowers. However, being ‘natural’ does not necessarily mean they are safe to take. 1 Taking HMs may not be suitable for children: as for all medicines. The NHS recommends that parents ask a paediatrician or pharmacist before giving a HM to their children. 1 Purpose To investigate HM use in the paediatric population (0–18 years). Material and methods We conducted a prospective study, using a questionnaire delivered to parents waiting in a paediatric practice office. Questions concerned: most used HMs, general opinion about HMs and practical experience (as to their efficacy and side effects). Results 92 questionnaires have been collected to date. 90.2% of those interviewed were mothers. 78.3% affirmed the use of HMs for their children. HMs were mostly used for cough and cold syndromes. 76.9% of mothers bought HMs less than 5 times a year. HMs were mostly suggested by the paediatrician (49.2%) or pharmacist (47.7%), and less frequently by the seller in the herbalist’s shop (10.8%) or by the natural health practitioner (6.2%). 63.6% considered HMs use as an alternative medicine, 50.6% as effective as conventional medicine and 44.4% affirmed that the efficacy HMs depends on the product. 83.8% considered HMs less dangerous than conventional drugs. 84% of parents said that children did not develop side effects after HM administration. The majority of parents reported HM use to the doctor in the case of drug prescription (45.1%); 68.4% were aware of interactions of HMs with other substances. Conclusion Even if this subject is relevant and our data show that HMs are frequently used in children, overall information are lacking in the literature. Hence one of our purposes would be to implement our study, extending it to a more widespread population. References and/or acknowledgements 1. National Health Service, Herbal Medicines http://www.nhs.uk/Conditions/herbal-medicines/Pages/Introduction.aspx (accessed 15 October 2016). No conflict of interest

Hyaluronic acid (HA) is a naturally-occurring glycosaminoglycan and a major component of the extracellular matrix. Low levels of the hyaluronic acid receptor CD44 are found on the surface of epithelial, hematopoietic, and neuronal cells; it is overexpressed in many cancer cells, and in particular in tumor-initiating cells. HA has recently attracted considerable interest in the field of developing drug delivery systems, having been used, as such or encapsulated in different types of nanoassembly, as ligand to prepare nano-platforms for actively targeting drugs, genes, and diagnostic agents. This review describes recent progress made with the several chemical strategies adopted to synthesize conjugates and prepare novel delivery systems with improved behaviors.

Paclitaxel has been found to be very effective against several human cancers; one of the major problems with its use is its poor solubility, which makes necessary its solubilization with excipients that can determine allergic reactions often severe. The aim of this study is to develop highly water-soluble prodrugs of paclitaxel. For this purpose we prepared a series of new paclitaxel–poly(ethylene glycol) (PEG) conjugates that were characterized and evaluated for their in vitro stability and cytotoxicity. In particular, in order to modulate the release of paclitaxel from prodrugs, we prepared different compounds introducing PEG in the drug C2′ and/or C7 positions via ester or carbamate linkage. The conjugates were obtained in high purity and good yield. The carbamate prodrugs were highly stable in different media, while the compounds obtained linking PEG at C2′ position through an ester bond showed lower stability. Finally, the cytotoxic activity of the conjugates was evaluated on two cancer cell lines and the results showed that all the derivatives had a reduced cytotoxicity compared to that of paclitaxel.

PEGylation is one of the most successful strategies to improve the delivery of therapeutic molecules such as proteins, macromolecular carriers, small drugs, oligonucleotides, and other biomolecules. PEGylation increase the size and molecular weight of conjugated biomolecules and improves their pharmacokinetics and pharmacodinamics by increasing water solubility, protecting from enzymatic degradation, reducing renal clearance and limiting immunogenic and antigenic reactions. PEGylated molecules show increased half-life, decreased plasma clearance, and different biodistribution, in comparison with non-PEGylated counterparts. These features appear to be very useful for therapeutic proteins, since the high stability and very low immunogenicity of PEGylated proteins result in sustained clinical response with minimal dose and less frequent administration. PEGylation of liposomes improves not only the stability and circulation time, but also the 'passive' targeting ability on tumoral tissues, through a process known as the enhanced permeation retention effect, able to improve the therapeutic effects and reduce the toxicity of encapsulated drug. The molecular weight, shape, reactivity, specificity, and type of bond of PEG moiety are crucial in determining the effect on PEGylated molecules and, at present, researchers have the chance to select among tens of PEG derivatives and PEG conjugation technologies, in order to design the best PEGylation strategy for each particular application. The aim of the present review will be to elucidate the principles of PEGylation chemistry and to describe the already marketed PEGylated proteins and liposomes by focusing our attention to some enlightening examples of how this technology could dramatically influence the clinical application of therapeutic biomolecules.

Study Type – Therapy (case control) Level of Evidence 3b OBLECTIVES To assess in a phase II pharmacokinetic study whether different pH levels, dilution volumes and exposure times affect intracellular bioavailability and systemic absorption of gemcitabine. SUBJECTS AND METHODS Six arms of three patients each with a non-muscle-invasive bladder cancer (NMIBC) were planned to receive six combinations of two different dilution volumes (50 mL vs 100 mL), two pH levels (2.5–3.5 vs 5.5) and two exposure times (1 h vs 2 h) of the study drug. Blood samples were taken before, during and 1 h after drug instillation. Cold biopsy specimens from the exophytic tumor, its base of implant and a macroscopically healthy mucosa were taken during transurethral resection. High-pressure liquid chromatography/high-resolution mass spectrometry (HPLC/HRMSn) analysis of plasma and tissue samples was used to determine concentrations of gemcitabine (dFdC) and its inactive metabolite (dFdU). RESULTS The arm at pH 5.5 in 50 mL was withdrawn as 2000 mg dFdC are insoluble in these conditions. The different instillation conditions resulted in negligible plasma dFdC concentrations but significant differences in intracellular content and metabolism of dFdC. The lowest intratissue concentration of dFdC was detected in a 50 mL solution at a pH of 2.5–3.5 kept in the bladder for 1 h (standard arm). A pH 5.5 solution in 100 mL with a 2-h exposure favored the maximal intratumoral dFdC absorption which was 90 times higher than that recorded in the standard arm. CONCLUSIONS The most commonly reported administration scheme of gemcitabine produced the lowest tissue bioavailability of dFdC. Other combinations of pH, dilution volume and duration of instillation proved more advantageous and merit testing in clinical trials.

Oxaliplatin is a promising drug for cancer therapy and the oxaliplatin/5-fluorouracil/leucovorin (FOLFOX) regimen has become the standard adjuvant treatment for colorectal cancer. However, the oxaliplatin-induced neurotoxicity still represents a clinical problem leading to a discontinuation of the therapy. Many strategies have been proposed in order to manage the neurotoxicity, but their effect on antitumoral efficacy is still unclear. In this study, we investigated the effect of reduced glutathione administration on neurotoxicity, oxaliplatin pharmacokinetics, and platinum-DNA (Pt-DNA) adduct formation in patients affected by colorectal cancer treated with FOLFOX4 adjuvant regimen. Twenty-seven patients were randomized to receive GSH 1500 mg/m or saline solution before oxaliplatin infusion. Evaluation of neurotoxicity, pharmacokinetics of plasmatic total and ultrafiltered Pt, and determination of Pt-DNA adduct formation on white blood cells was performed during the 5th, 9th, and 12th cycles. At the end of all cycles of therapy, the patients in the GSH arm showed a statistically significant reduction of neurotoxicity (P=0.0037) compared with the placebo arm. There were no significant differences in the main pharmacokinetic parameters between the two arms except a lower area under the plasma concentration-time curve and a smaller apparent steady-state volume of distribution (Vss) when GSH was coadministered. This difference can be explained by the natural function of GSH in the detoxification of oxaliplatin and by its ability to remove the Pt bound to plasma proteins. The determination of Pt-DNA adduct formation shows no statistically significant differences between the two arms. In conclusion, this study indicates that coadministration of GSH is an effective strategy to reduce the oxaliplatin-induced neurotoxicity without impairing neither the pharmacokinetics of oxaliplatin, nor the Pt-DNA adduct formation.

The study determined pharmacokinetic parameters, toxicity profile and preliminary clinical activity of gemcitabine administered i.v. at different infusion rates in patients with a range of solid tumors.Twenty patients were enrolled for both pharmacokinetic and clinical studies. Gemcitabine 300 mg/m(2) was administered during 1 h, 2 h or 3 h, and as a conventional dose of 1000 mg/m(2) during 30 min infusion. Administration was on days 1, 8 and 15 every 4 weeks.Patients were randomly assigned to one of the four arms. After 30 min infusion of 1000 mg/m(2) gemcitabine the plasma concentration remained above the saturation level of 10-20 microM, whereas after 1, 2 or 3 h infusion 300 mg/m(2) gemcitabine it remained below the saturation level for most of the time (being in the range 2.5-10 microM). Gemcitabine triphosphate was determined in the four arms in white blood cells; for infusion times from 0.5 to 3 h there was a progressive enhancement of gemcitabine triphosphate levels. In all evaluable patients the toxicity was mild, myelosuppression being the main toxicity. No grade 3 or 4 toxicities occurred. Clinical response was similar in patients receiving 300 mg/m(2) gemcitabine in 2 and 3 h and in the 1000 mg/m(2) arm.300 mg/m(2) gemcitabine during 3 h infusion produced the highest accumulation of gemcitabine triphosphate. Thus, to achieve the highest possible gemcitabine triphosphate level, prolonged infusion time would appear to be more important than a high dose administered as a short infusion. However, there was no substantial difference in toxicity or antitumoral activity in the all different patient groups.

New iodoacetamide derivatives, containing a dodecyl or a squalenyl moiety, were synthesized. The effect of these new thiol-reacting molecules was studied on two mutants of Alicyclobacillus acidocaldarius squalene-hopene cyclase constructed especially for this purpose. In the quintuple mutant, all five cysteine residues of the enzyme are substituted with serine; in the sextuple mutant, this quintuple substitution is accompanied by the substitution of aspartate D376, located at the enzyme's active site, with a cysteine. N-Dodecyliodoacetamide had little activity toward either mutant, whereas N-squalenyliodoacetamide showed a stronger effect on the sextuple than on the quintuple mutant, as expected.

The function of squalene-hopene cyclase from Alicyclobacillus acidocaldarius was studied by labelling critical cysteine residues of the enzyme, either native or inserted by site-directed mutagenesis, with different thiol-reacting molecules. The access of the substrate to the active centre cavity through a nonpolar channel that contains a narrow constriction harbouring a cysteine residue (C435) was probed by labelling experiments on both a C435S mutant, lacking C435 of the channel constriction, and a C25S/C50S/C455S/C537S mutant, bearing C435 as the only cysteine residue. Labelling experiments with tritiated 3-carboxy-4-nitrophenyl-dithio-1,1',2-trisnorsqualene (CNDT-squalene) showed that the cysteine residue at the channel constriction was covalently modified by the squalene-like inhibitor. Time-dependent inactivation of the C25S/C50S/C455S/C537S mutant by a number of squalene analogues and other agents with thiol-modifying activity suggested that modifying C435 caused the obstruction of the channel constriction thus blocking access of the substrate to the active site. The tryptic fragment comprising C435 of the quadruple mutant labelled with the most effective inhibitor had the expected altered molecular mass, as determined by LC-ESI-MS measurements. The arrangement of the substrate in the active site cavity was studied by using thiol reagents as probes in labelling experiments with the double mutant D376C/C435S in which D376, supposedly the substrate-protonating residue, was substituted by cysteine. The inhibitory effect was evaluated in terms of the reduced ability to cyclize oxidosqualene, as the mutant is unable to catalyse the reaction of squalene to hopene. Among the inhibitors tested, the substrate analogue squalene-maleimide proved to be a very effective time-dependent inhibitor.

trans-Vinyldioxidosqualene and beta-hydroxysulfide derivatives were synthesized stereospecifically and evaluated as inhibitors of animal and yeast oxidosqualene cyclases. Only trans-vinyldioxidosqualene and 2,3-epoxy-vinyl-beta-hydroxysulfides, having the reactive function at crucial positions 14,15 and 18,19, were active as inhibitors of animal and yeast cyclases. (14-trans)-28-Methylidene-2,3: 14,15-dioxidoundecanorsqualene 27 was the most potent inhibitor of the series of pig liver cyclase, with an IC50 of 0.4 microM, and it behaved also as the most active time-dependent inhibitor of the animal enzyme.

To evaluate, for the first time, the mitomycin C (MMC) pharmacokinetics during intravesical hyperthermia treatment based on conductive heat and the stability and recovery of the drug at the end of the instillation period.Eleven patients with recurrent intermediate-risk superficial transitional cell carcinoma of the bladder were treated weekly for six cycles with intravesical MMC (40 mg MMC in 50 ml) in local hyperthermia (45 °C) with Unithermia(®) system. Each instillation lasted 45 min, with the solution being replaced after the first 22 min. The MMC recovery at the end of the two instillation period and the plasmatic pharmacokinetics of MMC were evaluated by high-pressure liquid chromatography.Nine patients completed all the six planned cycles, whereas two patients missed the last cycle because of allergic reactions. No other systemic toxicity was observed, and the local toxicities were mild. Median MMC concentration in the instillation residual solution decreases from the initial 0.8 to 0.22 mg/ml for the 0-22-min instillation period and to 0.38 mg/ml for the 22-45-min instillation period; the median percentage of MMC recovered after instillation was 66.2 and 99.6, respectively. In all patients, MMC plasmatic C max resulted considerably lower than the toxic threshold (400 ng/ml).The MMC is stable during the instillation, and its absorption occurs mainly during the first minutes of the treatment. The plasmatic MMC concentration is always well below the threshold level for myelosuppression, as confirmed by the total lack of hematological toxicity evidenced by the patients. In order to evaluate the efficacy of the treatment performed with UniThermia(®) in reducing the disease recurrence rate in short- and long-term follow-up, we are currently carrying out a clinical multicentric study involving a larger number of patients.

The enzyme properties and location of essential functional groups of solubilized oxidosqualene cyclase of Candida albicans have been studied. We show that the C. albicans enzyme is much more heat-labile compared with Saccharomyces cerevisiae and rat liver cyclases, requires a histidyl residue for enzyme activity, contains an essential thiol residue either close to or in the active site and exhibits a carbocationic mechanism for catalysis, as the enzyme-bound substrate protects the enzyme from inactivation by a site-directed inactivator.

EC-145, under development by Endocyte, is a conjugate composed of desacetylvinblastine monohydrazide linked through a peptide spacer to the targeting moiety folic acid, for the potential intravenous treatment of folate receptor-overexpressing tumors, in particular ovarian and lung cancers. In vitro studies demonstrated that EC-145 selectively binds to cells that overexpress the folate receptor, causing dose-dependent cytotoxicity. Furthermore, coincubation of the KB human nasopharyngeal carcinoma cell line with EC-145 and doxorubicin resulted in synergistic antitumor activity. Experiments in mouse tumor xenograft models have confirmed the potency of EC-145 and the curative effects of the drug conjugate were demonstrated in an aggressive lymphoma xenograft model. In a phase I clinical trial in patients with advanced or metastatic solid tumors, adverse events were generally of moderate severity with the most frequent being fatigue, constipation and peripheral sensory neuropathy. Preliminary data from a phase II clinical trial in patients with advanced ovarian cancer demonstrated that third- or fourth-line treatment with EC-145 yielded better disease control than second- or third-line liposomal doxorubicin. Coadministration of EC-145 and liposomal doxorubicin produced a statistically significant increase in progression-free survival over standard therapy in patients with platinum-resistant ovarian cancer. Phase III clinical trials are expected to confirm these promising results.

The synthesis of (E)- and (Z)-29-methylidyne-2,3-oxidosqualene derivatives is described starting from the C22 and C17 squalene aldehyde monobromohydrins. The conversion was achieved by means of a Wittig reaction, followed by desilylation of the terminal acetylene. For trisubstituted 1,3-enynes, preliminary alkylation with a suitable allyl bromide was performed. A new procedure for the synthesis of squalene aldehyde C27, C22 and C17 monobromohydrins is also described. Some of the new compounds behaved as inhibitors of pig liver and yeast oxidosqualene cyclase and were time-dependent inhibitors of the animal enzyme.

This multicenter pharmacokinetic study evaluated the pharmacokinetics and toxicity of the combination of pegylated liposomal doxorubicin (PLD) and vinorelbine (VNR) in patients with recurrent ovarian cancer, when administered in opposing sequences.Eighteen heavily pre-treated ovarian cancer patients received treatment with PLD 30 mg/m2 and VNR 30 mg/m2 every 3 weeks for 6 cycles, 9 being given the PLD-VNR sequence vs. 9 the VNR-PLD sequence.The VNR AUCtot and plasma levels were considerably higher with the PLD-VNR sequence and VNR clearance, clearly related to Kel, was about half that occurring with VNR-PLD. Toxicity was generally mild and reversible. In both arms, a sound correlation was found between VNR AUCtot and absolute neutrophil count decrease. A possible correlation between hematological toxicity and the 2 opposing administration sequences was also shown.The higher VNR AUCtot and plasma levels during the elimination phase with the PLD-VNR sequence may be related to a P-gp membrane glycoprotein inhibition by PLD vescicles which, in turn, may influence the plasma level of the associated VNR. PLD-VNR, producing a higher plasma level and very mild toxicity, may be considered the preferred sequence.

In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants. These results suggested a possible interaction between these two enzymes. In this study, we present evidence that Erg27p interacts with Erg7p, facilitating the association of Erg7p with lipid particles (LPs) and preventing digestion of Erg7p both in the endoplasmic reticulum (ER) and LPs. We demonstrate that Erg27p is required for oxidosqualene cyclase (Erg7p) activity in LPs, and that Erg27p co-immunoprecipitates with Erg7p in LPs but not in microsomal fractions. While Erg27p is essentially a component of the ER, it can also be detected in LPs. In erg27 strains, a truncated Erg7p mislocalizes to microsomes. Restoration of Erg7p enzyme activity and LPs localization was achieved in an erg27 strain transformed with a plasmid containing a wild-type ERG27 allele. We suggest that the physical interaction of Erg27p with Erg7p is an essential regulatory tool in yeast sterol biosynthesis.

Various (1E,3E)- and (1Z,3E)-conjugated methylthio derivatives of oxidosqualene (OS) and conjugated and non-conjugated phenylthio derivatives of OS were obtained. These compounds, designed as inhibitors of pig liver and Saccharomyces cerevisiae 2,3-oxidosqualene-lanosterol cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene-hopene cyclase (SHC) (EC 5.4.99.-), contain the reactive function adjacent to carbons involved in the formation of the third and the fourth cycle during OS cyclization. All the new compounds are inhibitors of OSC and SHC, with various degrees of selectivity. The conjugated methylthio derivatives behaved as potent inhibitors of S. cerevisiae OSC, whereas most of the phenylthio derivatives were especially active toward SHC.

Cycloartenol synthase from Arabidopsis thaliana and lanosterol synthase from Trypanosoma cruzi and Pneumocystis carinii were expressed in yeast, and their subcellular distribution in the expressing cells was compared. Determination of enzymatic (oxidosqualene cyclase, OSC) activity and SDS-PAGE analysis of subcellular fractions proved that enzymes from T. cruzi and A. thaliana have high affinity for lipid particles, a subcellular compartment rich in triacylglycerols, and steryl esters, harboring several enzymes of lipid metabolism. In lipid particles of strains expressing the P. carinii enzyme, neither OSC activity nor the electrophoretic band at the appropriate M.W. were detected. Microsomes from the three expressing strains retained some OSC activity. Affinity of enzymes from A. thaliana and T. cruzi for lipid particles is similar to that of OSC of Saccharomyces cerevisiae, which is mainly located in this compartment. A different distribution of OSC in yeast cells suggests that they differ in some structural features critical for the interaction with the surface of lipid particles. Computer analysis supports the hypothesis of the structural difference since OSC from S. cerevisiae, A. thaliana, and T. cruzi lack or contain only one transmembrane spanning domain (a structural feature that makes a protein poorly inclined to associate with lipid particles), whereas OSC from P. carinii possesses six transmembrane domains. In the strain expressing cycloartenol synthase from A. thaliana, the accumulation of lipid particles largely exceeded that of the other strains.

The function of squalene-hopene cyclase from Alicyclobacillus acidocaldarius was studied by labelling critical cysteine residues of the enzyme, either native or inserted by site-directed mutagenesis, with different thiol-reacting molecules. The access of the substrate to the active centre cavity through a nonpolar channel that contains a narrow constriction harbouring a cysteine residue (C435) was probed by labelling experiments on both a C435S mutant, lacking C435 of the channel constriction, and a C25S/C50S/C455S/C537S mutant, bearing C435 as the only cysteine residue. Labelling experiments with tritiated 3-carboxy-4-nitrophenyl-dithio-1,1',2-trisnorsqualene (CNDT-squalene) showed that the cysteine residue at the channel constriction was covalently modified by the squalene-like inhibitor. Time-dependent inactivation of the C25S/C50S/C455S/C537S mutant by a number of squalene analogues and other agents with thiol-modifying activity suggested that modifying C435 caused the obstruction of the channel constriction thus blocking access of the substrate to the active site. The tryptic fragment comprising C435 of the quadruple mutant labelled with the most effective inhibitor had the expected altered molecular mass, as determined by LC-ESI-MS measurements. The arrangement of the substrate in the active site cavity was studied by using thiol reagents as probes in labelling experiments with the double mutant D376C/C435S in which D376, supposedly the substrate-protonating residue, was substituted by cysteine. The inhibitory effect was evaluated in terms of the reduced ability to cyclize oxidosqualene, as the mutant is unable to catalyse the reaction of squalene to hopene. Among the inhibitors tested, the substrate analogue squalene-maleimide proved to be a very effective time-dependent inhibitor.

Various vinyl sulfide and ketene dithioacetal derivatives of truncated 2,3-oxidosqualene were developed. These compounds, having the reactive functions at positions C-2, C-15 and C-19 of the squalene skeleton, were studied as inhibitors of pig liver and Saccharomyces cerevisiae oxidosqualene cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene hopene cyclase (SHC) (EC 5.4.99.-). They contain one or two sulfur atoms in alpha-skeletal position to carbons considered to be cationic during enzymatic cyclization of the substrate and should strongly interact with enzyme nucleophiles of the active site. Most of the new compounds are inhibitors of the OSC and of SHC, with various degrees of selectivity. The methylthiovinyl derivative, having the reactive group at position 19, was the most potent and selective inhibitor of the series toward S. cerevisiae OSC, with a concentration inhibiting 500% of the activity of 50 nM, while toward the animal enzyme it was 20 times less potent. These results could offer new insight for the design of antifungal drugs.

The inhibition of squalene-hopene cyclase (SHC) (E.C. 5.4.99.-), an enzyme of bacterial membranes catalyzing the formation of pentacyclic sterol-like triterpenes, was studied by using different classes of compounds originally developed as inhibitors of oxidosqualene cyclase (OSC) (E.C. 5.4.99.7), the enzyme of eukaryotes responsible for the formation of tetracyclic precursors of sterols. The mechanism of cyclization of squalene by SHC, beginning with a protonation of the 2,3 double bond by an acidic residue of the enzyme, followed by a series of electrophilic additions of the carbocationic intermediates to the double bonds, is similar to the mechanism of cyclization of 2,3-oxidosqualene by OSC. The inhibitors studied included: (i) analogs of the carbocationic intermediates formed during cyclization, such as aza-analogs of squalene and 2,3-oxidosqualene; (ii) affinity-labeling inhibitors bearing a methylidene reactive group; and (iii) vinyldioxidosqualenes and vinylsulfide derivatives of the substrates. Comparison of the results obtained with the two enzymes, SHC and OSC, showed that many of the most effective inhibitors of OSC were also able to inhibit SHC, while some derivatives acted as specific inhibitors. Differences could be easily explained on the basis of the different substrate specificity of the two enzymes.

19-Azasqualene-2,3-epoxide was more inhibitory than the corresponding N-oxide against 2,3-oxidosqualene cyclase (OSC) solubilized from Saccharomyces cerevisiae (IC50 7+/-2 and 25+/-5 microM, respectively). Both compounds showed a reversible, noncompetitive-type inhibition on solubilized OSC. Different inhibitory properties between the compounds were especially evident when measuring [14C]acetate incorporation into nonsaponifiable lipids extracted from treated cells. In cells treated with 19-azasqualene-2,3-epoxide at 30 microM, the radioactivity associated with the oxidosqualene fraction, which was negligible in the controls, rose to over 40% of the nonsaponifiable lipids, whereas it remained at a slightly appreciable level in cells treated with the N-oxide derivative under the same conditions. 19-Azasqualene-2,3-epoxide was also more effective than the N-oxide as a cell growth inhibitor (minimal concentration of compound needed to inhibit yeast growth: 45 and100 microM, respectively). The two inhibitors underwent different metabolic fates in the yeast: while 19-azasqualene-2,3-epoxide did not undergo any transformation, its N-oxide was actively reduced to the corresponding amine in whole and in "ultrasonically stimulated" cells. The N-oxide reductases responsible for this transformation appear to be largely confined within the microsomal fractions and require NADPH for their activity. A possible relationship between the inhibitory properties of the two compounds and their metabolic fates is discussed.

Background Rheumatoid arthritis (RA) is an autoimmune disorder, affecting 1% of the population, characterised by pain, joint swelling and progressive destruction of joint tissue. EULAR (European League Against Rheumatism) recommends the use of Tumour Necrosis Factor alpha antagonists (anti-TNFα) if methotrexate or Disease Modifying Antirheumatic Drugs fail. Anti-TNFα treatment imposes a significant financial burden on hospital budgets. Purpose To perform a pharmacoeconomic investigation in the Piedmont region (Italy) to identify the cost of the illness RA. To analyse the payer’s and societal perspectives, investigating direct costs associated with health care use and indirect costs related to productivity loss. Materials and Methods A multidisciplinary group, rheumatologists, hospital pharmacists and pharmacoeconomists, was established to perform a pharmacoeconomic evaluation of the direct and indirect costs of RA, by a systematic literature review. Afterward, we plan a perspective, observational, multicentre, cost-effectiveness analysis of RA biological drugs, involving 100 patients. Each patient will be recorded, every three months for one year, through personal data, disease duration and characterization, systemic manifestations and comorbidities, prescribed biological medicines. A questionnaire will be submitted, in order to assess direct and indirect costs. Results 40 existing pharmacoeconomic evaluations were critically appraised: the overall mean costs of RA amounted to about €15,000 per year, while the direct annual costs of RA were on average about €4,000. The greatest burden of RA costs was the indirect costs. From a societal perspective the superior clinical outcomes achieved with anti-TNFα are worth their higher costs. The most favourable incremental cost-effectiveness ratio was for etanercept compared to methotrexate. Conclusions The cost-effectiveness of an intervention depends on the maximum the decision makers are willing to pay for an extra unit of health effect. It should be considered that treatments with anti-TNFα, in a societal perspective, decrease the use of health resources and increase productivity. No conflict of interest.

The serine proteinase catalyzed hydrolysis of N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p- nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C. The results are consistent with the minimum three-step catalytic mechanism. The acylation step is rate limiting for human (Lys 77 species) and porcine plasmin, and for bovine beta-trypsin, the deacylation rate being limiting, on the other hand, for human and bovine alpha-, beta- and gamma-thrombin. Moreover the M(r) 33,000 species of human urokinase and the neuraminidase-treated porcine pancreatic beta-kallikrein-B do not catalyze the hydrolysis of the tripeptide. According to the specificity properties of the serine proteinases considered. Eoc-D-Phe- Pro-azaLys-ONp shows the characteristics of a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin.

Kinetics of bovine β-trypsin (trypsin) with the N α -( N,N -dimethylcarbamoyl)-α-aza-lysine p -nitrophenyl ester (Dmc-azaLys-ONp) was obtained at pH 6.2 and 21.0°C. Dmc-azaLys-ONp shows the characteristics of an optimal active site titrant in that it (i) gives titrations in a short time, (ii) is a stable and soluble compound with a stoichiometric reaction that is easily and directly detectable, and (iii) allows titrations over a wide range of enzyme concentration. Moreover, the three-dimensional structure of the trypsin · N α -( N - N -dimethylcarbamoyl)-α-aza-lysine acyl · enzyme adduct has been solved by X-ray crystallography at 2.0 A resolution ( R = 0.145). The Dmc-azaLys moiety of the active site titrant is sited in the serine proteinase reaction center, and is covalently linked to the OG atom of the Ser 195 catalytic residue.

The abilities of 22,23-epoxy-2-aza-2,3-dihydrosqualene and the corresponding N-oxide, 22,23-epoxy-2-aza-2,3-dihydrosqualene-N-oxide, to inhibit sterol biosynthesis were studied in microsomes and cells of Saccharomyces cerevisiae and Candida albicans. 22,23-Epoxy-2-aza-2,3-dihydrosqualene, which differs from the other inhibitor only in lacking oxygen at position 2, exhibited higher inhibitory properties in all preparations tested. The different levels of effectiveness of the two azasqualene derivatives were evident mostly in microsomes from S. cerevisiae (the 50 inhibitory concentrations of the 2-aza derivative and the corresponding N-oxide on oxidosqualene cyclase were 30 and 120 microM respectively) and in cell cultures of the same strain (1 order of magnitude separated the inhibitory activities of the two compounds on sterol biosynthesis). A possible explanation for the differences between 22,23-epoxy-2-aza-2,3-dihydrosqualene and the corresponding N-oxide arose from the study of their metabolic fates in vivo and in vitro. While the 2-aza derivative did not undergo any transformation, the N-oxide compound was actively reduced to the corresponding amine in microsomes and in cells of both yeast strains. 22,23-Epoxy-2-aza-2,3-dihydrosqualene-N-oxide seems to behave as a proinhibitor of sterol biosynthesis, becoming active only after transformation into the active form 22,23-epoxy-2-aza-2,3-dihydrosqualene.

13012 Background: The PTX-PLD association is a promising schedule for recurrent head/neck cancer. Their pk behavior could be dependent not only on PTX excipient (polyethoxylated castor oil) interference, but even on different i.v. administration intervals between the two drugs. This study evaluated any possible administration interval-dependent pk interaction, when PLD infusion started 0, 1, 3, 12 or 24 h after PTX infusion end. Methods: 25 patients, affected by recurrent cisplatin pre-treated squamous cell head/neck cancer, were randomized to receive PTX 80 mg/m2 q1w and PLD 12.5 mg/m2 q2w at administration intervals of 0, 1, 3, 12 or 24 h. Pk parameters were evaluated during the first course by non-compartmental analysis, while statistical analysis was performed by non-parametric Kruskal Wallis test. Results: Median PK parameters are reported in the table . PTX pk profile is strongly affected by PLD administration. PTX total exposure is highly reduced, with a consequent increase in Cltot; this alteration is totally due to Kel modifications. On the other side, no statistically significant interactions affected PLD pk parameters. Some in vitro experiments indicate that PLD is able to partially absorb PTX, driving to PTX plasmatic concentration reduction, when PLD is administered at 0–1 h intervals. Conclusions: PLD liposomal components seem to be able to entrap PTX, therefore reducing PTX plasmatic concentrations; so, it is very important to choose the ideal administration interval. In order to avoid pk interaction, the i.v. administration interval between PTX and PLD had to be 3 h at least. For shorter intervals, patients could be underexposed to PTX, with lesser clinical efficacy. [Table: see text] No significant financial relationships to disclose.

2559 Background: Neurotoxicity is a common OXA toxicity in FOLFOX4 regimen for patients with advanced colorectal cancer. Recently, Cascinu et al. (JCO 2002; 20: 3478–3483) provided evidence that GSH reduces the OXA-induced neurotoxicity, but GSH influence on the formation of Pt-DNA adducts still remains unknown. This study evaluated the effect of GSH addition on OXA pk and on Pt-DNA adducts formation Table of Contents Methods: 28 patients were given twelve FOLFOX4 courses and randomized to receive either GSH 1,500 mg/m2 or normal saline solution (placebo) before OXA iv infusion. OXA pk and Pt-DNA adducts formation were evaluated at courses 5, 9 and 12. Total and ultrafiltered platinum were analyzed by atomic adsorption, Pt-DNA adducts in leukocytes (as model for tumour tissue) by adsorptive stripping voltammetry. Pk analysis were done by non-compartmental analysis, statistical analysis by non-parametric Mann-Whitney test. Results: Median total and ultrafiltered platinum median Cmax and AUCtot values were comparable to previously reported ones, being higher in the placebo arm, due to a moderate reduction of platinum clearance. The formation of Pt-DNA adducts was more pronounced in GSH arm (median value 20.3 Pt atoms/106 nucleotides vs. 5.7 Pt atoms/106 nucleotides), even not statistically significant. Conclusions: The addition of GSH to FOLFOX4 regimen is able to reduce the OXA-induced neurotoxicity, without affecting either the OXA pk behaviour or the formation of Pt-DNA adducts, without modifying FOLFOX4 clinical efficacy. [Table: see text] No significant financial relationships to disclose.