Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.

Hospital effluents management: Chemical, physical, microbiological risks and legislation in different countries.

Hospital wastewater (HWW) can contain hazardous substances, such as pharmaceutical residues, chemical hazardous substances, pathogens and radioisotopes. Due to these substances, hospital wastewater can represent a chemical, biological and physical risk for public and environmental health. In particular, several studies demonstrate that the main effects of these substances can't be neutralised by wastewater treatment plants (WWTPs). These substances can be found in a wide range of concentrations due to the size of a hospital, the bed density, number of inpatients and outpatients, the number and the type of wards, the number and types of services, the country and the season. Some hazardous substances produced in hospital facilities have a regulatory status and are treated like waste and are disposed of accordingly (i.e., dental amalgam and medications). Legislation is quite homogeneous for these substances in all industrial countries. Problems that have emerged in the last decade concern substances and microorganisms that don't have a regulatory status, such as antibiotic residues, drugs and specific pathogens. At a global level, guidelines exist for treatment methods for these effluents, but legislation in all major industrial countries don't contain limitations on these parameters. Therefore, a monitoring system is necessary for these effluents as well as for substances and pathogens, as these elements can represent a risk to the environment and public health.

Phase I and pharmacodynamic study of high-dose NGR-hTNF in patients with refractory solid tumours.

BACKGROUND: NGR-hTNF exploits the peptide asparagine-glycine-arginine (NGR) for selectively targeting tumour necrosis factor (TNF) to CD13-overexpressing tumour vessels. Maximum-tolerated dose (MTD) of NGR-hTNF was previously established at 45 µg m(-2) as 1-h infusion, with dose-limiting toxicity being grade 3 infusion-related reactions. We explored further dose escalation by slowing infusion rate (2-h) and using premedication (paracetamol). METHODS: Four patients entered each of 12 dose levels (n=48; 60-325 µg m(-2)). Pharmacokinetics, soluble TNF receptors (sTNF-R1/sTNF-R2), and volume transfer constant (K(trans)) by dynamic imaging (dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)) were assessed pre- and post-treatment. RESULTS: Common related toxicity included grade 1/2 chills (58%). Maximum-tolerated dose was not reached. Both C(max) (P<0.0001) and area under the plasma concentration-time curve (P=0.0001) increased proportionally with dose. Post-treatment levels of sTNF-R2 peaked significantly higher than sTNF-R1 (P<0.0001). Changes in sTNF-Rs, however, did not differ across dose levels, suggesting a plateau effect in shedding kinetics. As best response, 12/41 evaluable patients (29%) had stable disease. By DCE-MRI, 28/37 assessed patients (76%) had reduced post-treatment K(trans) values (P<0.0001), which inversely correlated with NGR-hTNF C(max) (P=0.03) and baseline K(trans) values (P<0.0001). Lower sTNF-R2 levels and greater K(trans) decreases after first cycle were associated with improved survival. CONCLUSION: asparagine-glycine-arginine-hTNF can be safely escalated at doses higher than MTD and induces low receptors shedding and early antivascular effects.

Phase II study of pemetrexed and carboplatin plus bevacizumab as first-line therapy in malignant pleural mesothelioma.

BACKGROUND: The aim of this open label phase II study (NCT00407459) was to assess the activity of the vascular endothelial growth factor (VEGF) inhibitor bevacizumab combined with pemetrexed and carboplatin in patients with previously untreated, unresectable malignant pleural mesothelioma (MPM). METHODS: Eligible patients received pemetrexed 500 mg m(-2), carboplatin area under the plasma concentration-time curve (AUC) 5 mg ml(-1) per minute and bevacizumab 15 mg kg(-1), administered intravenously every 21 days for six cycles, followed by maintenance bevacizumab. The primary end point of the study was progression-free survival (PFS). A 50% improvement in median PFS in comparison with standard pemetrexed/platinum combinations (from 6 to 9 months) was postulated. RESULTS: Seventy-six patients were evaluable for analysis. A partial response was achieved in 26 cases (34.2%, 95% CI 23.7-46.0%). Forty-four (57.9%, 95% CI 46.0-69.1%) had stable disease. Median PFS and overall survival were 6.9 and 15.3 months, respectively. Haematological and non-haematological toxicities were generally mild; however, some severe adverse events were reported, including grade 3-4 fatigue in 8% and bowel perforation in 4% of patients. Three toxic deaths occurred. CONCLUSION: The primary end point of the trial was not reached. However, due to the limitation of a non-randomised phase II design, further data are needed before drawing any definite conclusion on the role of bevacizumab in MPM.

Preclinical emergence of vandetanib as a potent antitumour agent in mesothelioma: molecular mechanisms underlying its synergistic interaction with pemetrexed and carboplatin.

BACKGROUND: Although pemetrexed, a potent thymidylate synthase (TS) inhibitor, enhances the cytoytoxic effect of platinum compounds against malignant pleural mesothelioma (MPM), novel combinations with effective targeted therapies are warranted. To this end, the current study evaluates new targeted agents and their pharmacological interaction with carboplatin-pemetrexed in human MPM cell lines. METHODS: We treated H2052, H2452, H28 and MSTO-211H cells with carboplatin, pemetrexed and targeted compounds (gefitinib, erlotinib, sorafenib, vandetanib, enzastaurin and ZM447439) and evaluated the modulation of pivotal pathways in drug activity and cancer cell proliferation. RESULTS: Vandetanib emerged as the compound with the most potent cytotoxic activity, which interacted synergistically with carboplatin and pemetrexed. Drug combinations blocked Akt phosphorylation and increased apoptosis. Vandetanib significantly downregulated epidermal growth factor receptor (EGFR)/Erk/Akt phosphorylation as well as E2F-1 mRNA and TS mRNA/protein levels. Moreover, pemetrexed decreased Akt phosphorylation and expression of DNA repair genes. Finally, most MPM samples displayed detectable levels of EGFR and TS, the variability of which could be used for patients' stratification in future trials with vandetanib-pemetrexed-carboplatin combination. CONCLUSION: Vandetanib markedly enhances pemetrexed-carboplatin activity against human MPM cells. Induction of apoptosis, modulation of EGFR/Akt/Erk phosphorylation and expression of key determinants for pemetrexed and carboplatin activity contribute to this synergistic interaction, and, together with the expression of these determinants in MPM samples, warrant further clinical investigation.

Spontaneous preference for visual cues of animacy in naïve domestic chicks: The case of speed changes.

Animacy perception arises in human adults from motion cues implying an internal energy source to the moving object. The internal energy of the object is often represented by a change in speed. The same features cause preferential attention in infants. We investigated whether speed changes affecting adults' animacy ratings elicit spontaneous social preferences in visually-naïve chicks. Human observers evaluated the similarity between the movement of a red blob stimulus and that of a living creature. The stimulus entered the screen and moved along the azimuth; halfway through its trajectory it could either continue to move at a constant speed or linearly increase in speed. The average speed, the distance covered and the overall motion duration were kept constant. Animacy ratings of humans were higher for accelerating stimuli (Exp. 1). Naïve chicks were then tested for their spontaneous preference for approaching the stimulus moving at a constant speed and trajectory or an identical stimulus, which suddenly accelerated and then decelerated again to the original speed. Chicks showed a significant preference for the 'speed-change stimulus' (Exp. 2). Two additional controls (Exp. 3 and 4) showed that matching the variability of the control 'speed-constant' stimulus to that of the 'speed-change stimulus' did not alter chicks' preference for the latter. Chicks' preference was suppressed by adding two occluders on both displays, positioned along the stimulus trajectory in such a way to occlude the moment of the speed change (Exp. 5). This confirms that, for chicks to show a preference, the moments of speed change need to be visible. Finally, chicks' preference extended to stimuli displaying a direction change, another motion cue eliciting animacy perception in human observers, if the speed- and direction-profile were consistent with each other and resembled what expected for biological entities that invert their motion direction (Exp. 6). Overall, this is the first demonstration of social predispositions for speed changes in any naïve model or non-human animal, indicating the presence of an attentional filter tuned toward one of the general properties of animate creatures. The similarity with human data suggests a phylogenetically old mechanism shared between vertebrates. Finally, the paradigm developed here provides ground for future investigations of the neural basis of these phenomena.

Detection of pathogenic Campylobacter, E. coli O157:H7 and Salmonella spp. in wastewater by PCR assay.

The aim of this study was the evaluation of the occurrence of pathogenic Campylobacter, Escherichia coli O157:H7, E. coli virulence genes and Salmonella spp. in different wastewater treatment plants (WWTPs) using a method based on an enrichment step and PCR. This method was sensitive enough to detect low levels (∼2 CFU100 ml(-1) of raw sewage) of all the investigated pathogens. In the WWTP samples, E. coli O157:H7 DNA and the eae gene were never found, but 33 % of influents and effluents exhibited amplicons corresponding to Shiga-like toxin I. Twenty-five percent of the influent and 8 % of the effluent exhibited the presence of Shiga-like toxin II. Campylobacter jejuni and C. coli DNA were identified in 50 and 25 % of the influents and in 8 and 25 % of the effluents, respectively. Salmonella spp. DNA was present in all the samples. Considering the results obtained, the method tested here offers a reliable and expeditious tool for evaluating the efficiency of the effluent treatment in order to mitigate contamination risk. Influent contamination by Salmonella spp. and Campylobacter spp. provides indirect information about their circulation; moreover, their presence in effluents underlines the role of WWTPs in the contamination of the receiving surface waters, which affects public health directly or indirectly.

A randomized trial for the treatment of high-grade soft-tissue sarcomas of the extremities: preliminary observations.

A new trial for evaluating the effectiveness of adjuvant chemotherapy in high-grade soft-tissue sarcomas of the extremities in adult patients is presented. All patients after local treatment were randomized into two arms, one without further therapy and the other to receive adjuvant chemotherapy (Adriamycin [Farmitalia-Carlo Erba, Milan, Italy], 450 mg/m2). The preliminary results of the study are reported at a median observation period of 27.6 months. Of the 59 patients who entered the study, 79.1% in the chemotherapy group are without sign of disease, whereas the corresponding figure in the nonadjuvant chemotherapy group is 54.3%. The difference between the two groups is statistically significant (P less than .005, log rank test). These preliminary observations encourage continuation of the study.

Selectivity in capillary electrophoresis: the use of proteins.

Proteins, by their very diverse nature, provide a wide variety of options for generating selectivity in capillary electrophoresis (CE). Their use in different modes of CE will be considered in this review. Proteins added in solution to the background electrolyte allow separations to be made in a similar fashion to other electrokinetic chromatography methods, e.g., micellar separations. Alternatively, different immobilization schemes can be used to secure proteins within the capillary; these have included capillary electrochromatography with the protein grafted onto a silica support, or immobilization of the protein within a gel structure. Compounds varying in size from small inorganic ions to biopolymers may be bound by proteins. There is the potential for any sort of intermolecular interaction to play a role in the binding process (e.g., hydrophobic interactions, electrostatic interactions, etc.). Very specific high-affinity binding often occurs, but also there is often weaker, non-selective binding. Frequently the interactions of chiral compounds with proteins are stereoselective. Obtaining chiral selectivity has been one of the main applications of protein selectors in CE, and this use will be emphasized here in a discussion structured by type of protein. As well as utilizing the selectivity of proteins to develop separations, the role of CE in investigating ligand-protein interactions will be emphasized.

Immobilized penicillin G acylase as reactor and chiral selector in liquid chromatography.

In this paper, the use of penicillin G acylase (PGA) as a biocatalyst and as a chiral selector is described. Penicillin G-acylase is an interesting enzyme used in the manufacture of semisynthetic antibiotics and, in particular, in the production of 6-APA by hydrolysis of penicillin G. Five PGA-based HPLC columns have been prepared by using two different silica supports by employing two immobilization methods, namely "in situ" and "in batch". The effects of the immobilization techniques and of different silica pore size on the catalytic properties of the enzyme as well as the applicability of the PGA-bonded stationary phases as chiral selectors for a number of chiral drugs have been investigated. The HPLC columns based on immobilized PGA combine the hydrolytic activity and the chiral recognition properties of PGA, therefore they have been used for the development of a combined reaction-separation system for chiral and achiral substrates.

Validation of a RP-LC method for the simultaneous determination of isoniazid, pyrazinamide and rifampicin in a pharmaceutical formulation.

A simple and accurate liquid chromatographic method was developed and validated for estimation of isoniazid (ISN), pyrazinamide (PYR) and rifampicin (RIF) in combined dosage forms. Drugs were chromatographed on a reverse phase C18 column using a mobile phase gradient and monitored at the corresponding maximum of each compounds. Peaks were identified with retention time as compared with standards and confirmed with characteristic spectra using diode-array detector. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method does not require any specific sample preparation except the use of a guard column. The method is linear (r(2)>0.999), precise (RSD%: 0.50% for ISN, 0.12% for PYR and 0.98% for RIF), accurate (overall average recovery yields: 98.55% for ISN, 98.51 for PYR and 98.56% for RIF) and selective. Due to its simplicity and accuracy the method is suitable for routine quality control analysis of antitubercolosis combination dosage form.

Design of chiral LC separations for calcium antagonists on alpha 1-acid glycoprotein and ovomucoid columns.

Three chiral calcium antagonist drugs, gallopamil and two dihydropyridine derivatives, have been successfully separated within short retention times using both the alpha 1-acid glycoprotein chiral stationary phase (Chiral-AGP) and the ovomucoid column (Ultron ES-OVM). Aqueous buffer at defined pH is modified by the addition of an organic component, in order to modulate the retention properties of each system. Optimization of pH and organic modifier is carried out using the modified simplex method, with Kaiser's peak separation function as a criterion. The influence of pH and percentage of organic modifier on retention, selectivity, resolution and column performance are discussed for the two dihydropyridines analysed on Chiral-AGP and Ultron ES-OVM stationary phases. A new method is proposed as a new chiral system suitability test for these protein-based phases, utilizing a racemic mixture of closely eluting verapamil enantiomers as a probe.

[Evaluation of the changes in glycosylated hemoglobin in a sample of hospitalized diabetic patients. Significance and limitations of this diagnostic method]. / Valutazione delle variazioni dell'emoglobina glicosilata in un campione di soggetti diabetici ospedalizzati. Significato e limiti di questa diagnostica.

Stable glycosylated hemoglobin measurement provides an index for plasma glucose control over an average three month period. It is not clear whether stable HbA1C can undergo rapid changes induced by short-term variations in glucose concentration. The present study was aimed at investigating whether variation of total HbA1C in response to changes in blood glucose, over a short period can be detected by routine methods of determination. Our result show that due to the sensitivity of the methods, stable HbA1C has to be considered only as an index of glucose control over long period of time.

[Clinical experience with a new antibiotic: cefmetazole]. / Esperienza clinica su un nuovo antibiotico: il cefmetazolo.

Cefmetazole is a new semisynthetic derivative with a broad antibacterial spectrum as shown by data from 34 research institutes in Japan. It has antibacterial action against various species of Gram+ and Gram- bacteria and is extremely stable for beta-lactamase because it has a 7-alpha-methoxy group. In addition, it has excellent antibacterial activity against indole-positive Proteus strains against which conventional Cephalosporins are ineffective. Results of clinical trials carried out on urinary tract, respiratory tract and cutaneous infections are reported. A total of 30 patients were examined; the route of administration was intravenous, the most frequent daily-dose was 2 g. Clinical and bacteriological effectiveness of cefmetazole was 80% with excellent activity against E. coli, Proteus, S. aureus and K. pneumoniae. No serious side effect was observed. The above results indicate that CMZ is largely effective and useful for infections due to Gram+ cocci and Gram- bacilli.

[Therapeutic use of glutathione]. / Utilizzo terapeutico del glutatione.

Liver pathology is very frequent in western countries. The numerous therapeutic approaches suggested so far for the prevention or limitation of liver damage in the course of hepatic diseases have often had disappointing results. Considering that at least part of the liver injury is caused by free radicals, the authors have tried an antioxidant, glutathione, as a hepatoprotective agent. Although particularly brilliant results were not achieved, clinical conditions and laboratory findings were improved in hall patients treated with glutathione.

[Evaluation in experimental animals of a mathematical model of foreseeing variations in values of stable glycosylated hemoglobin]. / Valutazione sull'animale da esperimento di un modello matematico per la previsione delle variazioni dei valori di emoglobina glicosilata stabile.

Assay of glycosylated hemoglobin provides reliable information on metabolic control in diabetes mellitus over a period of about 90 days. This is why it is currently used as a parameter of blood glucose control in diabetic patients. However, at present little is known about the kinetics of stable glycosylated hemoglobin variations as a result of circadian changes in blood glucose level. The authors describe a mathematical model which allows to foresee glycosylated hemoglobin variability as a result of alterations of blood glucose equilibrium.

[Characteristics of voluntarily discharged patients: some reflections and a proposal]. / Caratteristiche dei pazienti con dimissione volontaria dall'ospedale: alcune riflessioni ed una proposta.

This study compares the characteristics of 492 patients discharged against medical advice from the Istituto Dermopatico dell'Immacolata, a Research Hospital (335 beds) in Rome specialized in Dermatology, Vascular Surgery and Plastic Surgery between 1995 and 1998, with those of 43,110 control patients discharged with physicians' approval according to a case-control model. In the multivariate analysis, male gender (OR 1.65; 95% CI 1.37-1.98) and residence in Rome area (OR 1.22; 95% CI 1.02-1.47) increased the odds of discharge against medical advice. The odds of such discharge decreased with increasing age (OR 0.994 per year; 95% CI 0.990-0.999). The proportion of patients discharged against medical advice decreased from 1.4% in 1995 to 0.4% in 1998. The average length of stay decreased from 1995 to 1998, with the exception of the Plastic Surgery unit. A retrospective review of the charts of 45 patients discharged against medical advice (AMA) within 48 hours from admission ascertained some of the stated reasons for discharge. Personal and family problems or refusal of treatment were reported for 35% of the patients. Of the 25 patients who gave no reasons, 11 asked again for hospitalization and 5 of these were rehospitalized by IDI within 10 days. This study also identified that the information on the medical record of patients who left against medical advice was generally poor. A standardized form for AMA discharges, including patient's understanding of the diagnosis, treatment, alternative therapies, consequences of refusing treatment and stated reasons for leaving against medical advice, might be of benefit to patients, physicians and hospital managers.

The impact of capillary electrophoresis in drug analysis and bioanalysis.

Capillary electrophoresis (CE) has penetrated a wide variety of areas of separation science during the last ten years. The relative strengths and weaknesses of this technique are here overviewed, by citing the most recent literature and updated Medline search, keeping an eye on technology news, as technology and the development of CE and its applicability progress in parallel. The issues discussed include micellar electrokinetic chromatography (MEKC), which permits non-ionic solutes to be solubilized and separated. Enantioselective applications in CE exploit addition to the run buffer of cyclodextrins, macrocyclic antibiotics and proteins for analysis of chiral drugs and the state of the art of this field will be emphasized. CE has significant potential for drug metabolism studies, especially coupled with MS for high sensitivity detection and structural characterisation. Strategies for the analysis of drugs and metabolites in body fluids include on-capillary methods of sample concentration and single-step analyses with direct injection of body fluids on-column, where detection techniques other than conventional UV are essential to achieve adequate sensitivity. The potential contribution of the hybrid technique of capillary electrochromatography (CEC), which couples the separating power of reversed-phase HPLC and the high efficiency of CE, has recently attracted growing interest and is therefore discussed. Finally, validation issues which are peculiar to CE are illustrated.

Comparison of drug binding sites on rat and human serum albumins using immobilized-protein stationary phases as a tool for the selection of suitable animal models in pharmacological studies.

Immobilized serum albumins (rat and human) HPLC stationary phases have been used to investigate and to compare the binding sites of a number of analytes of pharmaceutical interest assessing the retention time as the chromatographic parameter which correlates the percentage of drug-binding of the corresponding free protein. Anti-inflammatory drugs with different molecular structure were chromatographed and the k' values were determined with a mobile phase based on 100 mM sodium phosphate buffer (pH 6.9) containing 6% n-propanol (v/v). The effect of the addition to the mobile phase of octanoic, decanoic and dodecanoic acids on the k' values enabled to elucidate the site of interaction between the solutes and the two albumins. The results indicate that there are some structural differences in the binding sites of the two albumins and also some quantitative differences with respect to the extent of drug-binding. Moreover the different stereoselective behaviour of the two albumins was studied by analysing some chiral anti-inflammatory drugs much as aryl-propionic acids.

Vinorelbine in pemetrexed-pretreated patients with malignant pleural mesothelioma.

BACKGROUND: Pemetrexed-platinum chemotherapy is the standard first-line treatment of unresectable malignant pleural mesothelioma (MPM). At progression, patients are generally selected to experimental trials, when available, or, in every-day clinical practice, they are offered second-line chemotherapy. The optimal treatment has not yet been defined. The aim of this retrospective, single-center study was to evaluate the activity and toxicity of vinorelbine administered to a consecutive series of pemetrexed-pretreated MPM patients. METHODS: Vinorelbine 25 mg/m(2) was administered intravenously as a single agent on days 1, 8 every three weeks, either as second-line (2L) or further-line (>2L) therapy. Treatment was repeated for a maximum of 6 cycles, until progression, or unacceptable toxicity. RESULTS: Fifty-nine patients were included in this analysis. Vinorelbine was given to 34 patients as 2L, and to 25 as > 2L treatment. The median age was 69 years (range 45-80). Forty-two patients (71.2%) had a good EORTC prognostic score. Partial response was observed in 9 (15.2%) cases, stable disease in 20 (33.9%). The overall disease control rate (DCR) was 49.1%. Median progression-free survival (PFS) and overall survival (OS) were 2.3 and 6.2 months, respectively. ECOG performance status (PS) (HR(0 vs. 1-2) 0.50; 95%CI: 0.3-0.8; p = 0.014) and PFS ≥ 6 months following first-line (FL) chemotherapy (HR(FL-PFS>6 ms vs. <6 ms) 0.50; 95%CI: 0.3-0.9; p = 0.031) were significantly associated to OS in multivariate analysis. No difference was observed in terms of DCR, PFS, and OS in relation to age, histology, sex, line of vinorelbine therapy, or response to FL treatment. Hematological toxicity was acceptable, with grade 3/4 neutropenia occurring in 5 (8.4%) patients, and there were no cases of febrile neutropenia. The main non-hematological toxicities were grade 2 fatigue in 17 (28.8%) and constipation in 7 (11.8%) patients. CONCLUSIONS: Vinorelbine was moderately active in pemetrexed-pretreated MPM patients, with an acceptable toxicity profile, particularly in patients with ECOG-PS0 and FL-PFS ≥ 6 months.