Biomedical subjects

S Orlandini

Publications and source records attributed to S Orlandini.

At least 19 recordsLinked to original sources

PubMed2006

Competitive bond breaking in floppy molecular trimers: HeNeH and HeNeH- calculations.

Calculations for the J=0 bound states associated with the two title molecular systems are carried out using distributed Gaussian function approach and using the sum of two-body potential approximation to describe the overall interactions. The results yield one bound state for the neutral trimer and three bound states for the anionic triatom. The relative values of dissociation energies place the first dissociation threshold at the H emission for the neutral complex and at the He emission for the corresponding anion. The general spatial properties of the various bound states are analyzed on both systems.

Journal Article↗

PubMed2005

Determination of stability constant values of flurbiprofen-cyclodextrin complexes using different techniques.

Three new experimental approaches for calculating the stability constant (K(st)) of complexes of flurbiprofen with natural beta-cyclodextrin (betaCyd) and the hydroxyethyl- (HEbetaCyd) and the methyl- (Me betaCyd) derivatives were tested and compared to the classic phase-solubility procedure: (a) the membrane permeation technique through a lipophilic synthetic membrane permeable to the drug but not to the Cyd molecules, by analysing the permeation profiles with a non-linear least-squares method; (b) the affinity capillary electrophoresis (ACE) technique, where K(st) were calculated from the relationship between Cyd concentration in solution and drug electrophoretic mobility, using three different linear plotting methods; (c) the molecular modeling technique, based on the relationship between the docking energies and the experimental K(st) values. The study allowed evaluation of the advantages and limits of each examined method, providing a useful guide for the choice of the most suitable one depending on the kind of host-guest system to be investigated. The K(st) values obtained with the various techniques were rather different, probably due to the very different experimental conditions required by each one. However, all the methods indicated the methyl-derivative as the most powerful complexing agent for the drug, showing the general trend: K(st)(Me betaCyd)>>K(st)(HEbetaCyd)>K(st)(betaCyd). Only in the case of the ACE method was an inversion of the trend found between HEbetaCyd and betaCyd; this was probably due to the lower molecular weight of the natural Cyd, which, in this case, became more important in determining the complex electrophoretic mobility than the different affinity of the drug for these two Cyds.

Chemistry, Pharmaceutical↗

PubMed2004

Micellar electrokinetic chromatography for the simultaneous determination of ketorolac tromethamine and its impurities. Multivariate optimization and validation.

A simple, fast and selective micellar electrokinetic chromatographic (MEKC) method for the simultaneous assay of ketorolac tromethamine and its known related impurities (1-hydroxy analog of ketorolac, 1-keto analog of ketorolac and decarboxylated ketorolac), in both drug substance and coated tablets, is described. The compounds were detected at 323 nm, and flufenamic acid (FL) and tolmetin (TL) were chosen as internal standards to quantify ketorolac tromethamine and impurities, respectively. The multivariate optimization of the experimental conditions was carried out by means of the response surface study, considering as responses the resolution values and analysis time. The optimized background electrolyte (BGE) consisted of a mixture of 13 mM boric acid and phosphoric acid, adjusted to pH 9.1 with 1 M sodium hydroxide, containing 73 mM sodium dodecyl sulfate (SDS). Optimal temperature and voltage were 30 degrees C and 27 kV. Applying these conditions, all compounds were resolved in about 6 min. The related substances could be quantified up to the 0.1% (w/w) level. Validation was performed, either for drug substances and drug product, evaluating selectivity, robustness, linearity and range, precision, accuracy, detection and quantitation limits and system suitability.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2003

How experimental design can improve the validation process. Studies in pharmaceutical analysis.

A critical discussion about the possibility of improving the method validation process by means of experimental design is presented. The reported multivariate strategies concern the evaluation of the performance parameters robustness and intermediate precision, and the optimisation of bias and repeatability. In particular, accuracy and precision improvement constitutes a special subset of experimental design in which the bias and the relative standard deviation of the assay are optimised. D-optimal design was used in order to plan experiments for this aim. The analytical methods considered were capillary electrophoresis, HPLC, adsorptive stripping voltammetry and differential pulse polarography. All methods were applied to real pharmaceutical analysis problems.

Adsorption↗

PubMed2003

Optimization of dissolution test precision for a ketoprofen oral extended-release product.

An example of application of experimental design methodologies to the set up of dissolution test conditions for a new ketoprofen oral extended-release dosage form is presented. The aim of the work was to find the best experimental conditions, using a USP apparatus 2 (paddle), for maximizing the method precision as degree of repeatability. The considered factors mainly influencing the dissolution test results were pH and volume of dissolution medium, and paddle stirring speed. Two distinct 4-run Plackett-Burman designs were carried out: one at gastric and the other at intestinal pH values. Each run was performed in triplicate in order to calculate the standard deviations of the drug dissolution efficiency at 60 and 120 min, selected as responses to be minimized. Optimum conditions to carry out the dissolution test were: 900 ml volume of dissolution medium and 70 rpm paddle stirring speed for both environments and pH 1 and 5.5, for the gastric and intestinal environment, respectively.

Administration, Oral↗

PubMed2002

Optimization and validation of a CZE method for rufloxacin hydrochloride determination in coated tablets.

A simple and rapid capillary electrophoresis method with UV detection was developed and validated for the determination of rufloxacin hydrochloride in coated tablets. An experimental design strategy (Doehlert design and desirability function) allowed the analytical parameters to be simultaneously optimized in order to determine rufloxacin hydrochloride with high peak area/migration time ratio, good efficiency and short analysis time. Optimized analyses were run using boric acid 0.10 M adjusted to pH 8.8 as BGE and setting voltage and temperature at 18 kV and 27 degrees C, respectively. Pefloxacin mesylate was used as internal standard and run time was about three minutes. The method was validated for the drug substance and the drug product according to the ICH3 guidelines. Robustness was tested by experimental design using an eight-run Plackett-Burman matrix.

Anti-Infective Agents↗

PubMed2001

Successful xenografting of cryopreserved primary pancreatic cancers.

In order to assess the suitability of cryopreserved neoplastic tissues for xenografting into nude (nu/nu) mice, we compared the take rate in 28 samples of pancreatic ductal carcinoma. Eleven fresh samples were implanted in nu/nu mice, and 17 were frozen in cryopreserving solution and implanted at a later time. All samples were examined for the presence of neoplastic tissue in cryostat sections. A total of 15 tumors grew in the animals; five from the freshly implanted samples and ten from those cryopreserved. Ten xenografted tumors were characterized for alterations in p53, K-ras, and p16 genes, which were found in six, eight, and nine cases, respectively. Our results demonstrate that the take rate for xenografting is comparable between cryopreserved and fresh tissue samples. The procedure allows for the exchange of tumor material between institutions and permits the establishment of centralized facilities for the storage of an array of different primary tumor samples suitable for the production of in vivo models of cancers.

Adult↗

PubMed2001

Genetic profile of 22 pancreatic carcinoma cell lines. Analysis of K-ras, p53, p16 and DPC4/Smad4.

The K-ras, p53, p16 and DPC4 genes are among those most frequently altered in pancreatic ductal carcinoma. We analyzed 22 cell lines for alterations in these genes by direct sequence analysis and methylation-specific polymerase chain reaction. These cell lines showed mutations in K-ras and p53 at frequencies of 91% and 95%, respectively. Alterations in p16INK4a were found in all cases and included nine homozygous deletions, seven mutations and promoter methylation in six cases. Eight cell lines (36%) had an alteration of DPC4, including one mutation and seven homozygous deletions. The most typical mutational profile involved K-ras, p53, and p16INK4a, concurrently aberrated in 20 cases (91%). Eight cell lines had alterations in all four genes. Inactivation of DPC4 was always accompanied by alteration of all of the other three genes. This comprehensive data regarding the cumulative genetic alterations in pancreatic carcinoma cell lines will be of great value for studies involving drug sensitivity or resistance that may be associated with inactivation of a particular gene or molecular pathway.

Carcinoma, Pancreatic Ductal↗

PubMed2001

Optimisation and validation of a capillary electrophoresis method for the simultaneous determination of diazepam and otilonium bromide.

A simultaneous assay of diazepam and otilonium bromide in coated tablets by capillary zone electrophoresis (CZE) was developed. The influence of various parameters (voltage, temperature, buffer concentration and pH, ethanol percentage) on analysis time and on the theoretical plates of the two peaks was investigated by means of experimental design. A response surface study was carried out by means of a 27-run D-optimal matrix. The best background electrolyte was found to be 0.13 M, pH 2.9 Britton-Robinson buffer, containing 10% v/v ethanol. Other optimised parameters were voltage (30 kV) and temperature (30 degrees C). The UV detector for quantitation of otilonium bromide and diazepam was set at 280 nm and 230 nm, respectively. Procaine hydrochloride was used as internal standard and run time was less than five minutes. Validation was performed, for drug substance and drug product, according to ICH3 guidelines. For drug product the recovery for otilonium bromide and diazepam ranged from 98.3% to 101.2% and from 97.1% to 99.0%, respectively; the RSD values found for otilonium bromide and diazepam ranged from 2.4% to 3.0% and from 1.1% to 4.5%, respectively.

Calcium Channel Blockers↗

PubMed2001

Pancreatic tumours: molecular pathways implicated in ductal cancer are involved in ampullary but not in exocrine nonductal or endocrine tumorigenesis.

Alterations of K- ras, p53, p16 and DPC4/Smad4 characterize pancreatic ductal cancer (PDC). Reports of inactivation of these latter two genes in pancreatic endocrine tumours (PET) suggest that common molecular pathways are involved in the tumorigenesis of pancreatic exocrine and endocrine epithelia. We characterized 112 primary pancreatic tumours for alterations in p16 and DPC4 and immunohistochemical expression of DPC4. The cases included 34 PDC, 10 intraductal papillary-mucinous tumours (IPMT), 6 acinar carcinomas (PAC), 5 solid-pseudopapillary tumours (SPT), 16 ampulla of Vater cancers (AVC) and 41 PET. All tumours were also presently or previously analysed for K- ras and p53 mutations and allelic loss at 9p, 17p and 18q. Alterations in K- ras, p53, p16 and DPC4 were found in 82%, 53%, 38% and 9% of PDC, respectively and in 47%, 60%, 25% and 6% of AVC. Alterations in these genes were virtually absent in PET, PAC or SPT, while in IPMT only K- ras mutations were present (30%). Positive immunostaining confirmed the absence of DPC4 alterations in all IPMT, SPT, PAC and PET, while 47% of PDC and 38% of AVC were immunonegative. These data suggest that pancreatic exocrine and endocrine tumourigenesis involves different genetic targets and that among exocrine pancreatic neoplasms, only ductal and ampullary cancers share common molecular events.

Adenocarcinoma, Mucinous↗

PubMed2000

Allelotype of pancreatic acinar cell carcinoma.

Pancreatic acinar cell carcinoma (PAC) is a rare pancreatic tumor for which no information about chromosomal and gene anomalies is available. We performed genome-wide allelotyping of 9 PACs using DNA from 5 frozen and 4 paraffin-embedded samples and 76 PCR-amplified, chromosome-specific microsatellite markers. High degrees of allelic loss were found, with a mean fractional allelic loss of 0.33. Chromosomes 1p, 4q and 17p showed loss of heterozygosity in >70% of cases and chromosomes 11q, 13q, 15q and 16q, in 60% to 70% of cases. Chromosomes 3q, 6q, 8q, 18q and 21q showed loss in 50% to 60% of cases. All of the remaining chromosomes showed no or few allelic losses. The resulting allelotype of PAC is markedly different from that of either ductal or endocrine tumors of the pancreas, and the involvement of chromosomes 4q and 16q appears to be characteristic of this tumor type. High-resolution mapping of the 12 frequently altered chromosomes in 5 cases with 222 markers permitted subchromosomal localization of regions of consensus loss on 5 chromosomes, including 1p36.31, 3p25.2, 4q26-31.1, 15q15-22.1 and 16q21-q22.1. Our findings suggest that PAC tumorigenesis involves molecular pathways different from those occurring in more common pancreatic tumor types.

Adolescent↗

PubMed2000

Detection of bladder cancer by semi-automated microsatellite analysis of urine sediment.

Patients with transitional-cell carcinoma (TCC) require careful follow-up due to the high risk of recurrence. Cystoscopy and biopsy are reliable but invasive, while urine cytology is plagued by low sensitivity. It has recently been shown that allelic abnormalities detected by microsatellite analysis of DNA extracted from urine can be used to diagnose TCC with high reliability. As this analysis by classic techniques is unfeasible in a clinical setting, we performed a pilot study to determine the possibility of applying quick DNA extraction methods with laser detection and computer-based analysis of 15 fluorescently labeled PCR amplified microsatellites to detect molecular anomalies in urine sediment in 25 TCC follow-up patients. Of the eighteen cases with recurrent TCC, 14 (78%) were positive by the molecular test whereas only eight (44%) were detected by cytology. Of the seven patients with negative cystoscopy, one resulted positive by the molecular test and had recurrent TCC six-months later. Thus, this microsatellite analysis correctly predicted the clinical diagnosis in 84% (21/25) of cases, compared to 60% by cytology. The application of these semi-automated procedures allows the analysis of 18 samples with 15 markers in one day, encouraging a more expedient introduction into routine clinical use.

Aged↗

PubMed1999

The FHIT gene is expressed in pancreatic ductular cells and is altered in pancreatic cancers.

We examined 2 normal pancreata, 21 primary pancreatic ductal cancers, and 19 pancreatic cancer cell lines for Fhit expression and FHIT gene status. The normal pancreas expressed Fhit protein in the cytoplasm of ductular cells, whereas interlobular and larger ducts, acini, and insulae of Langerhans were negative. Fhit protein was detected by immunoblot assay in 11 pancreatic cancer cell lines; of the 8 cell lines lacking Fhit protein, 7 lacked FHIT mRNA and 1 showed an abnormally sized transcript. DNA from five of these eight cell lines showed homozygous loss of FHIT exon 5. In 8 of the 21 primary cancers, Fhit expression was detected by immunohistochemistry. Reverse transcription-PCR analysis of 6 of the 13 cases lacking Fhit showed normal-sized FHIT product in 3 cases and a mixture of normal and abnormal products in the other 3. Sequencing showed that abnormal bands were missing variable numbers of exons. Loss of microsatellite DNA markers internal to the FHIT gene was observed in 10 of 13 primary cancers lacking Fhit protein (homozygous in two cases) and in only 1 of the 8 cancers expressing Fhit protein. In nine primary cancers, four expressing and five lacking Fhit protein, it was possible to obtain pure cancer DNA by microdissection. Three of the five microdissected cases lacking Fhit protein exhibited homozygous deletion of FHIT exon 5. In conclusion, the lack of Fhit protein in pancreatic cancers correlated with absence or alteration of FHIT mRNA and was often associated with FHIT gene anomalies.

Acid Anhydride Hydrolases↗

PubMed1999

Immunohistochemical evidence suggests intrinsic regulatory activity of human eccrine sweat glands.

Immunohistochemistry of normal eccrine sweat glands was performed on paraffin sections of human skin. Immunoreactivity (ir) for neuron specific enolase, S100 protein (S100), regulatory peptides, nitric oxide synthase type I (NOS-I) and choline-acetyltransferase (ChAT) was found in small nerve bundles close to sweat glands. In the glands, secretory cells were labelled with anticytokeratin antibody. Using antibodies to S100, calcitonin gene-related peptide (CGRP) and substance P (SP) a specific distribution pattern was found in secretory cells. Granulated (dark) and parietal (clear) cells were immunopositive for CGRP, and S100 and SP, respectively. Immunoreactivity was diffuse in the cytoplasm for CGRP and S100, and peripheral for SP. Myoepithelial cells were not labelled. Electron microscopy revealed electron dense granules, probably containing peptide, in granulated cells. Using antibodies to NOS-I and ChAT, ir was exclusively found in myoepithelial cells. Immunoreactivity for the atrial natriuretic peptide was absent in sweat glands. These results provide evidence for the presence of both regulatory peptides involved in vasodilation and key enzymes for the synthesis of nitric oxide and acetylcholine in the secretory coil of human sweat glands. It is suggested that human sweat glands are capable of some intrinsic regulation in addition to that carried out by their nerve supply.

Adult↗

PubMed1998

Development and validation of a differential pulse polarographic method for quinolinic acid determination in human plasma and urine after solid-phase extraction: a chemometric approach.

A chemometric approach was applied for determining quinolinic acid in human plasma by differential pulse polarography after solid phase extraction. A fractional factorial design was used to examine the significant experimental variables for the peak height maximization. A Doehlert design, which allowed a sequential response surface methodology to be performed, was applied to the variables scan rate and drop size. The results indicated that the scan rate had the greatest effect on the response peak height. The linear range was extended from 8.52 x 10(-8) to 1.34 x 10(-5) M and the limit of detection was 2.9 x 10(-8) M. The validation process consisted of a pre-validation study followed by the main validation in the plasma matrix. The robustness and the intermediate precision were evaluated by means of experimental design. A 3(4)//9 screening symmetric matrix and a central composite design were used to optimize the solid phase extraction procedure of the analyte from human plasma using anion exchange cartridges. The goal was to select the best retention, wash and elution solvents and their volumes in order to maximize the extraction efficiency using as the response the polarographic peak height. An extraction efficiency of 90% was found. The method was also applied to the determination of quinolinic acid in urine and the mean concentration in human plasma and urine, was found to be 3.7 x 10(-7) and 4.9 x 10(-5) M respectively.

Chemistry Techniques, Analytical↗

PubMed1998

Molecular pathogenesis of sporadic duodenal cancer.

Whether duodenal adenocarcinoma should be considered as a gastrointestinal or as a peripancreatic cancer is a matter of debate, as is the opportunity and type of treatment. We investigated 12 such cancers for the genetic anomalies involved in the pathogenesis of gastrointestinal malignancies, including (a) those occurring in common-type cancers - allelic losses at chromosomes 3p, 5q, 17p and 18q, and Ki-ras and p53 alterations; and (b) those characteristic of mutator-phenotype cancers - microsatellite instability and TGF-betaRII gene mutations. We found Ki-ras and p53 mutations in five (42%) and eight cancers (67%), respectively; chromosome 3p, 5q, 17p and 18q allelic losses in two of nine (22%), six of ten (60%), six of nine (67%) and three of ten (30%) informative cancers, respectively. Finally, three cancers (25%) showed widespread microsatellite instability and two of them had a TGF-betaRII gene mutation. Our data suggest that duodenal cancers may arise from either of the two known pathogenetic molecular pathways of gastric and colorectal cancers. The majority of our cases were highly aggressive cancers with frequent chromosomal changes and p53 mutations as observed in the common-type gastrointestinal malignancies, while widespread subtle alterations characteristic of mutator-phenotype cancers occurred in a minority, which also showed a favourable long-term outcome.

Adult↗

PubMed1998

Chromosome 5 allelic losses are early events in tumours of the papilla of Vater and occur at sites similar to those of gastric cancer.

During our studies of DNA fingerprinting of tumours of the pancreas and papilla (ampulla) of Vater, using arbitrarily primed polymerase chain reaction (AP-PCR), we noticed two bands showing a decreased intensity in six of ten ampullary tumours with respect to matched normal tissues. Those bands were both assigned to chromosome 5. Such a finding was somewhat in contrast with the reportedly low frequency of APC gene mutations in ampullary cancers, located at chromosome 5q21, and suggested that loci different from that of APC might be the target of chromosome 5 allelic losses (LOH) in these tumours. Therefore, we analysed chromosome 5 LOH in a panel of 27 ampullary tumours, including eight adenomas, four early- and 15 advanced-stage cancers, using 16 PCR-amplified CA microsatellite polymorphic markers spanning the entire chromosome. Nineteen cases (70%) showed LOH, and the interstitial deletions found in these tumours described two smallest common deleted regions, in which putative suppressor genes might reside. They were at 5q13.3-q14 and at 5q23-q31 respectively, which correspond to those found in gastric tumours. In addition, the presence of 5q LOH in six of eight adenomas and in three of four early-stage cancers suggests that such phenomena occur at early stages of neoplastic progression of the ampullary epithelium.

Adenoma↗

PubMed1997

The human ALL-1/MLL/HRX antigen is predominantly localized in the nucleus of resting and proliferating peripheral blood mononuclear cells.

The ALL-1 gene is an important regulator of embryonal and hematopoietic development, and structural variants of the human gene generated by chromosomal translocations and other genomic alterations presumably act as oncogenes in the pathogenesis of acute leukemias and other hematological malignancies. Antisera against two different epitopes of the human ALL-1 protein (anti-ALL1-N and anti-ALL1-C) were produced. Both sera revealed indistinguishable patterns of antigen localization in human peripheral blood mononuclear cells (PBMCs). In resting PBMCs, the antigen was distributed in a speckled pattern across the nuclei, with an increased density at the nuclear envelope and the nuclear indentation. In mitotically stimulated PBMCs, the antigen surrounded the condensing chromosomes but did not colocalize with chromatin or the nuclear scaffold. The antigen is considered a marker for a novel nuclear subcompartment, a perichromosomal area termed the "chromosomal envelope." In Western blot experiments, the anti-ALL1-N serum reacted with a polypeptide corresponding to the expected full-length 430-kDa ALL-1 protein. Recombinant proteins representing the AT-hook and zinc binding subdomains of the ALL-1 protein interacted in vitro with a degenerate mixture of double-stranded oligodeoxynucleotides. Thus, the ALL-1 protein probably is a DNA-binding protein with both a sequence-unspecific (AT-hook) and a sequence-specific (zinc binding subdomains) double-stranded DNA binding mode.

Animals↗