Identification, synthesis and biological activity of alkyl-guanidine oligomers as potent antibacterial agents.

In the last two decades, the repertoire of clinically effective antibacterials is shrinking due to the rapidly increasing of multi-drug-resistant pathogenic bacteria. New chemical classes with innovative mode of action are required to prevent a return to the pre-antibiotic era. We have recently reported the identification of a series of linear guanidine derivatives and their antibacterial properties. A batch of a promising candidate for optimization studies (compound 1) turned out to be a mixture containing two unknown species with a better biological activity than the pure compound. This serendipitous discovery led us to investigate the chemical nature of the unknown components of the mixture. Through MS analysis coupled with design and synthesis we found that the components were spontaneously generated oligomers of the original compound. Preliminary biological evaluations eventually confirmed the broad-spectrum antibacterial activity of this new family of molecules. Interestingly the symmetric dimeric derivative (2) exhibited the best profile and it was selected as lead compound for further studies.

The epidemic of lung cancer in Tuscany (Italy): a joint analysis of male and female mortality by birth cohort.

Lung cancer epidemic among males and females was studied at small geographical level in Tuscany Region (Italy), about 3.5 million inhabitants over almost 30 years (1971-1999). The joint analysis of the space-time pattern of relative risk for a given cause on males and females was conducted specifying a series of Hierarchical Bayesian models. Goodness-of-fit, parsimony and robustness under misspecification were used to identify candidate models. We chose birth cohort as relevant time axis and restricted our attention to birth cohorts born between 1905 and 1940. We found very different pattern by gender: the epidemic curve among males had a peak for the birth cohort born around 1930, the younger cohorts being at lower risk. Among females the relative risk was rising almost linearly on the log scale, the younger birth cohorts being at higher risk. Both curves showed two accelerations corresponding to the post-war periods (1915-1929 and 1945-1959), assuming an average age at first exposure of 20 years old. The spatial distribution among the 287 municipalities investigated was characterized by high risks in all industrial areas in males, and limited to large towns in females. The shared spatial clustering component highlighted the historically developed part of the Tuscany Region.

Statistical methods for geographical surveillance in veterinary epidemiology.

Spatial clustering and cluster detection are statistical analysis developed to address relevant scientific hypothesis. The difficulty stays in the large number of alternative hypothesis due to the different mechanisms that could generate the anomalous cases aggregation. We review methods for marked point data (case/control) aimed to describe spatial intensity of disease risk, to test for randomness and to locate significant excesses. Bayesian Gaussian Spatial Exponential models are used to illustrate probabilistic aspects and the link with simpler non parametric tools are shown. We develop an informal guideline to the analysis and used data on faecal contamination and dog parasitic diseases in the city of Naples, Italy. Kernel density estimation resulted very sensitive to bandwidth choice and overemphasized localized excess, Ripley'K function and Cuzick-Edwards test were very consistent each other while the SatScan failed to detect excesses. The spatial range was around 600 meters and justifies several small clusters. Bayesian models were very powerful in reconstructing the phenomenon and allow inference on model parameters in good agreement with the non parametric analysis.

Statistical modelling of the spatial distribution of prevalence of Calicophoron daubneyi infection in sheep from central Italy.

Statistical modelling for Disease Mapping and Ecological Analysis is of particular importance in veterinary parasitology because environmental characteristics can affect parasite distribution. However, the main difficulties relate to the concentration of animal populations within farms, which contrasts to the study of wild animal populations. In the present paper we report the results of a cross-sectional coprological survey designed to study the presence and distribution of the rumen fluke Calicophoron daubneyi--which causes paramphistomosis, a snail borne disease--in pastured sheep living in the Latina province of central Italy. We show how techniques derived from human epidemiology can be used to study the spatial distribution of parasite infection in animals. We proposed a hierarchical Bayesian model with random terms for unstructured variability (heterogeneity) to account for local farm characteristics and spatially structure terms (clustering) to cope with medium-large scale environmental characteristics.

[Statistical models for spatial analysis in parasitology]. / Modelli statistici per l'analisi spaziale in parassitologia.

The simplest way to study the spatial pattern of a disease is the geographical representation of its cases (or some indicators of them) over a map. Maps based on raw data are generally "wrong" since they do not take into consideration for sampling errors. Indeed, the observed differences between areas (or points in the map) are not directly interpretable, as they derive from the composition of true, structural differences and of the noise deriving from the sampling process. This problem is well known in human epidemiology, and several solutions have been proposed to filter the signal from the noise. These statistical methods are usually referred to as Disease Mapping. In geographical analysis a first goal is to evaluate the statistical significance of the heterogeneity between areas (or points). If the test indicates rejection of the hypothesis of homogeneity the following task is to study the spatial pattern of the disease. The spatial variability of risk is usually decomposed into two terms: a spatially structured (clustering) and a non spatially structured (heterogeneity) one. The heterogeneity term reflects spatial variability due to intrinsic characteristics of the sampling units (e.g. igienic conditions of farms), while the clustering term models the association due to proximity between sampling units, that usually depends on ecological conditions that vary over the study area and that affect in similar way breedings that are close to each other. Hierarchical bayesian models are the main tool to make inference over the clustering and heterogeneity components. The results are based on the marginal posterior distributions of the parameters of the model, that are approximated by Monte Carlo Markov Chain methods. Different models can be defined depending on the terms that are considered, namely a model with only the clustering term, a model with only the heterogeneity term and a model where both are included. Model selection criteria based on a compromise between degree of complexity and goodness of fit are then needed to discriminate among them, because each specification has a different biological meaning. Our aim is to demonstrate that these techniques can be used to study the geographical distribution of a parasite infection. Our analyses are based on data collected in 142 farms of the province of Latina. In each breeding a fixed number of sheeps has been sampled (20) and checked for the presence of C. daubneyi. We have specified a Binomial model for the proportion of infected animals in each breeding. The heterogeneity component is modelled in a standard way, while we have used different prior specifications for the clustering term to show how they affect the results. When we use the usual specification also for clustering, the two models show a completely different spatial pattern of infection, probably because the intrinsic spatial structure of the clustering term tend to bias our inferences. The selection criterion indicates in this case the heterogeneity model as the "best" one. However, if we modify the prior so that a lower degree of spatial interaction is assumed, the clustering model is less complex and its goodness of fit better and it should be preferred.

Determination of levamisole in animal tissues using liquid chromatography with ultraviolet detection.

An efficient and sensitive liquid chromatographic method is described for the determination of the anthelminthic drug levamisole, in muscle, liver, kidney and fat of sheep, pigs and poultry, using thiabendazole as internal standard. Samples were extracted by homogenizing with chloroform, and were applied to Supelco Si solid-phase extraction columns and eluted with methanol. Chromatographic analysis was performed on a LiChrospher 60 RP-Select B column using methanol/ammonium acetate buffer 0.05 M (55/65, v/v) as mobile phase and reading at 220 nm. The quantification limit for the assay was 4 ng/g. Mean recoveries were about 84% for liver, 85% for kidney, 89% for muscle and 84% for fat. The assay has been used for statutory testing purposes.

High-performance liquid chromatographic assay of erythromycin from biological matrix using electrochemical or ultraviolet detection.

Two chromatographic methods were developed for the determination of erythromycin A (EA) residues in animal tissues (muscle, liver, kidney and fat of cattle, pigs and poultry) and cow's milk. In addition to a more traditional method using electrochemical detection, we developed an original alternative method based on UV detection at 236 nm, by pretreating to create a chromophore in the molecule. An internal standard was used with both methods to check the variability of the analytical system. Analysis times and performance were compared. The recovery of EA from various matrices was greater than 95%. For both methods the quantification limit for EA was 0.25 microgram ml-1 for plasma, 0.025 microgram g-1 for milk and 0.125 microgram g-1 for the other biological matrices. The methods can be used to check for EA residues in these matrices; in fact, the statutory maximum residue limits (MRLs) of EA are 0.4 microgram g-1 in muscle, kidney, liver and fat of beef cattle, sheep, pigs and poultry, and 0.04 microgram g-1 in cow's and sheep's milk.

[Socioeconomic inequalities in health in the Tuscany Longitudinal Study (SLTO): persistence and changes over time in overall mortality and selected causes (lung cancer, liver cirrhosis, AIDS and overdose)]. / Condizione socio-economica e disuguaglianze nella mortalità nello Studio Longitudinale Toscano (SLTo): persistenza ed evoluzione nel tempo (mortalità generale, tumore del polmone, cirrosi epatica), ed aspetti emergenti (AIDS e overdose).

We evaluate the persistence of social inequalities in overall mortality or mortality by causes (lung cancer, liver cirrhosis; AIDS and overdose) in the Tuscany Longitudinal Study (SLTo), a record linkage-study on the census population identified at the 1981 and 1991 censuses in Leghorn (1981: 175,741 subjects; 1991: 167,512 subjects), and 1991 in Florence (403,294 subjects), Central Italy. The census data allow an evaluation of socio-economic status of each subject, using variables such as education or occupation, or constructing indexes inclusive of deprivation indexes. Follow-up is from the census up to 1995 and for specific causes of death from 1987 (Leghorn) or 1991 (Florence). Estimates of risk are computed comparing rates of mortality among socio-economic groups, by means of poisson regression models, or by means of Standardized Mortality Ratios using the indirect method. The causes of deaths have been selected mainly because explained by known and strong determinants. As far as overall mortality, the results suggest the persistence of gradients by social class, more often negative among males. Mortality from lung cancer has a strong negative social class gradient among males, and a divergent gradient among women in the two towns, which is interpreted as the effect of a different prevalence of smoking by period, social class and sex. High RRs among lower socio-economic groups have been detected for liver cirrhosis mortality. Excess mortality of AIDS and overdose, an expression of the recent drug crisis, concentrates on the lower social strata and in young adults. Whereas mortality from AIDS has been detected among both sexes, deaths from overdose are occurring among males only. Being AIDS and overdose recent diseases, they stress the persistence of social inequalities over time.

[The Tuscany longitudinal study: mortality among selected causes in inner city of Florence and Leghorn]. / Studio Longitudinale Toscano: un'analisi per quartiere per le città di Firenze e Livorno.

The analysis of mortality in urban settings for the Cities of Florence (1991-95) and Leghorn (1987-95), based on data from the Tuscany Longitudinal Study, is reported in the present paper. The data came from a census-based cohort study, all residents at the census day 1981 (Leghorn) or 1991 (Florence) being enrolled and followed-up by automated procedures of record-linkage. The cause of death certificate had been eventually collected by the Regional Mortality Register. For each city, internally standardized mortality ratios (SMR) had been calculated by sub-urban areas (city sectors or wards). The analysis was restricted to age groups > 15 years to have interpretable results on socio-economic variables derived from census questionnaires. Bayesian estimates (Besag, York e Mollié) of mortality relative risks had been calculated to overcome extra-variability of SMRs. In the city of Florence two wards showed about 10% excess risk for overall mortality. In the city of Leghorn one sector was at higher risk while one showed a significant lower mortality. For both cities such risk gradients were still present after adjustment for deprivation index at individual level.

Near infrared transmittance analysis for the assay of solid pharmaceutical dosage forms.

The recent commercial availability of near infrared spectrometric instruments for the transmittance analysis of solids makes it possible to analyse solid drugs in their finished form. Application of the method to the control of the assay of the active ingredient in diphenhydramine tablets gave results comparable to those obtained in reflectance mode with whole and milled tablets.

Evaluation of release of antioxidant from high density polyethylene by planar chromatography.

Irganox 1330 (1,3,5-trimethyl-2,4,5-tris(3',5'-ditert-butyl)-4'-hydroxybenzyl)- benzene) (I), an alkylphenol compound used as antioxdiant in the production of high density polyethylene (HDPE), was the subject of this study. The quantity of I given up by polyethylene samples of different thickness to aqueous and lipophilic media simulating food was studied by planar chromatography (PC). The manner of antioxidant release was also investigated. The chromatographic method developed was found satisfactory for the quantification of I in the migration studies undertaken, and had quantification limits (12 ng/deposit) suitable for quality control of HDPE.

In vitro studies of simulated percutaneous absorption: influence of various enhancers in the release of clonazepam from 2-hydroxyethyl acetate patches.

A diffusion cell with an artificial membrane and the single-pass perfused rabbit ear were used to evaluate the percutaneous absorption of clonazepam from various 2-hydroxyethyl acetate (HEA) patches. The influence on drug permeation of the various type of enhancers (isopropylmyristate, lauryl alcohol, propylene glycol and water) in the patches was tested. A comparison between the two types of systems of percutaneous absorption of clonazepam has been done. The results showed that HEA patches produce controlled uniform drug release, modulated by the addition of enhancers.

Near infrared reflectance spectroscopy in the study of atopy. Part 3. Interactions between the skin and fomblins.

The near infrared reflectance spectra of the skin of atopic and normal subjects were compared after topical application of perfluorinated polyethers (fomblins) of different molecular weight and viscosity. It was possible to distinguish the two classes of subjects and the different fomblins applied, by principal components analysis.

Application of near-infrared reflectance spectrometry in the study of atopy. Part 1. Investigation of skin spectra.

An investigation into the existence of spectral differences and differences in response in terms of water and lipid content between normal and atopic skin is described. Since NIR radiation penetrates complex structured matrices down to a depth of 0.15-0.20 mm, it is evident that the method lends itself to spectral detection of skin components down to the deepest level. First the reproducibility of readings made with the instrument was tested and it was also checked whether the use of the probe caused changes in skin equilibrium due to occlusion. Analysis of the NIR spectra did not enable normal and atopic subjects to be distinguished unequivocally but provided important information on the use of NIR spectrometry in these subjects and insights into the stratum corneum. Although the responses of water and lipid structures could not be read directly from the spectra, it was possible to decompose the global spectral information into components by principal components analysis. It was possible to observe a fraction of variance associated in different ways with water.

Near-infrared reflectance spectrometry in the study of atopy. Part 2. Interactions between the skin and polyethylene glycol 400, isopropyl myristate and hydrogel.

An investigation into the existence of spectral differences and differences in response in terms of water and lipid content between normal and atopic skin after interaction with chemical agents is described. Three compounds were taken as models: a prevalently hydrophilic solvent (polyethylene glycol, PEG 400), a prevalently lipophilic solvent (isopropyl myristate) and a hydrophilic pharmaceutical (gel) used to promote contact in electrocardiography. Using principal component analysis it was possible to distinguish atopic and normal subjects by simple contact of the skin with chemical agents.

Planar chromatographic and liquid chromatographic analysis of thiamphenicol in bovine and human plasma.

A planar chromatographic (PC) method was developed to determine thiamphenicol in human and bovine plasma with florphenicol as internal standard. The performance of the method was compared with that of a liquid chromatographic method. Recovery of extraction method developed for plasma was 81.51 +/- 2.85%. Reproducibilities of the PC assay performed with various PC plates were also good.

Experimental design in the development of voltammetric method for the assay of omeprazole.

A multivariate strategy was used to optimize an adsorptive stripping voltammetric method for the determination of the antiulcer drug omeprazole. A 3/4 matrix was used for the variable screening while a central composite design was chosen in the subsequent step to evaluate the response surfaces. Simultaneous optimization of the response peak height (hp) and peak half width w1/2), the latter being a peak shape measure, was achieved. The factors accumulation time, pulse amplitude, scan rate and stirring rate were all found to be statistically significant for the response hp, while for the response w1/2 only the stirring rate was found to be significant. The optimized method shows a good linearity between peak height and analyte concentration in the concentration range from 8.33 x 10(-9) M to 1.42 x 10(-7) M with a LOD of 6.5 x 10(-9) M. The mean recovery of omeprazole in capsules was 101.9% with a SD of 2.04 (RSD = 200).

Application of near-infrared reflectance spectrometry to the analytical control of pharmaceuticals: ranitidine hydrochloride tablet production.

The possibility of applying near-infrared reflectance spectrometry to the control of the production cycle of ranitidine hydrochloride tablets was investigated. The results were good for the identification of ranitidine hydrochloride drug substance, mixtures for tablets, cores and coated tablets. The determination of the compound and of its water content also gave satisfactory results.

Design and optimization of the variables in the adsorptive stripping voltammetric determination of rufloxacin in tablets, human plasma and urine.

An adsorptive stripping voltammetric method with a hanging mercury drop electrode was developed for the determination of the fluoroquinolone rufloxacin in tablets, human plasma and urine. Measurements were obtained in differential pulse mode and a multivariate strategy was used to optimize the variables involved. Besides the independent effects of the variables, a strong interaction between scan rate and pulse duration has been found. Rufloxacin was analysed at concentrations between 1.7 x 10(-8) and 1.9 x 10(-7) M with a detection limit of 9.2 x 10(-9) M. Diluted tablet solutions and urine samples were analysed directly, while plasma samples needed an extraction procedure before voltammetric analysis. An improved HPLC procedure was used as comparative method.

Near infrared reflectance analysis: features and applications in pharmaceutical and biomedical analysis.

Growing interest has recently been expressed for the qualitative and quantitative analytical capabilities of Near Infrared Reflectance Spectroscopy (NIRS) in the measurement of physical and chemical parameters. The authors described the details of the method and its applications in the pharmaceutical and biomedical sectors.