Statins for cardiovascular prevention according to different strategies: a cost analysis.

Background: Several studies have shown that treatment with HMG-CoA reductase inhibitors (statins) can reduce coronary heart disease (CHD) rates. However, the cost effectiveness of statin treatment in the primary prevention of CHD has not been fully established. Objective: To estimate the costs of CHD prevention using statins in Switzerland according to different guidelines, over a 10-year period. Methods: The overall 10-year costs, costs of one CHD death averted, and of 1 year without CHD were computed for the European Society of Cardiology (ESC), the International Atherosclerosis Society (IAS), and the US Adult Treatment Panel III (ATP-III) guidelines. Sensitivity analysis was performed by varying number of CHD events prevented and costs of treatment. Results: Using an inflation rate of medical costs of 3%, a single yearly consultation, a single total cholesterol measurement per year, and a generic statin, the overall 10-year costs of the ESC, IAS, and ATP-III strategies were 2.2, 3.4, and 4.1 billion Swiss francs (SwF [SwF1 = $US0.97]). In this scenario, the average cost for 1 year of life gained was SwF352, SwF421, and SwF485 thousand, respectively, and it was always higher in women than in men. In men, the average cost for 1 year of life without CHD was SwF30.7, SwF42.5, and SwF51.9 thousand for the ESC, IAS, and ATP-III strategies, respectively, and decreased with age. Statin drug costs represented between 45% and 68% of the overall preventive cost. Changing the cost of statins, inflation rates, or number of fatal and non-fatal cases of CHD averted showed ESC guidelines to be the most cost effective. Conclusion: The cost of CHD prevention using statins depends on the guidelines used. The ESC guidelines appear to yield the lowest costs per year of life gained free of CHD.

Exploring unfrequent events with accelerated molecular dynamics simulations: from photochemistry to drug design

La meva incorporació al grup de recerca del Prof. McCammon (University of California San Diego) en qualitat d’investigador post doctoral amb una beca Beatriu de Pinós, va tenir lloc el passat 1 de desembre de 2010; on vaig dur a terme les meves tasques de recerca fins al darrer 1 d’abril de 2012. El Prof. McCammon és un referent mundial en l’aplicació de simulacions de dinàmica molecular (MD) en sistemes biològics d’interès humà. La contribució més important del Prof. McCammon en la simulació de sistemes biològics és el desenvolupament del mètode de dinàmiques moleculars accelerades (AMD). Les simulacions MD convencionals, les quals estan limitades a l’escala de temps del nanosegon (~10-9s), no son adients per l’estudi de sistemes biològics rellevants a escales de temps mes llargues (μs, ms...). AMD permet explorar fenòmens moleculars poc freqüents però que son clau per l’enteniment de molts sistemes biològics; fenòmens que no podrien ser observats d’un altre manera. Durant la meva estada a la “University of California San Diego”, vaig treballar en diferent aplicacions de les simulacions AMD, incloent fotoquímica i disseny de fàrmacs per ordinador. Concretament, primer vaig desenvolupar amb èxit una combinació dels mètodes AMD i simulacions Car-Parrinello per millorar l’exploració de camins de desactivació (interseccions còniques) en reaccions químiques fotoactivades. En segon lloc, vaig aplicar tècniques estadístiques (Replica Exchange) amb AMD en la descripció d’interaccions proteïna-lligand. Finalment, vaig dur a terme un estudi de disseny de fàrmacs per ordinador en la proteïna-G Rho (involucrada en el desenvolupament de càncer humà) combinant anàlisis estructurals i simulacions AMD. Els projectes en els quals he participat han estat publicats (o estan encara en procés de revisió) en diferents revistes científiques, i han estat presentats en diferents congressos internacionals. La memòria inclosa a continuació conté més detalls de cada projecte esmentat.

Quantification of typical antipsychotics in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A selective and sensitive method was developed for the simultaneous quantification of seven typical antipsychotic drugs (cis-chlorprothixene, flupentixol, haloperidol, levomepromazine, pipamperone, promazine and zuclopenthixol) in human plasma. Ultra-high performance liquid chromatography (UHPLC) was used for complete separation of the compounds in less than 4.5min on an Acquity UPLC BEH C18 column (2.1mm×50mm; 1.7μm), with a gradient elution of ammonium formate buffer pH 4.0 and acetonitrile at a flow rate of 400μl/min. Detection was performed on a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray ionization interface. A simple protein precipitation procedure with acetonitrile was used for sample preparation. Thanks to the use of stable isotope-labeled internal standards for all analytes, internal standard-normalized matrix effects were in the range of 92-108%. The method was fully validated to cover large concentration ranges of 0.2-90ng/ml for haloperidol, 0.5-90ng/ml for flupentixol, 1-450ng/ml for levomepromazine, promazine and zuclopenthixol and 2-900ng/ml for cis-chlorprothixene and pipamperone. Trueness (89.1-114.8%), repeatability (1.8-9.9%), intermediate precision (1.9-16.3%) and accuracy profiles (<30%) were in accordance with the latest international recommendations. The method was successfully used in our laboratory for routine quantification of more than 500 patient plasma samples for therapeutic drug monitoring. To the best of our knowledge, this is the first UHPLC-MS/MS method for the quantification of the studied drugs with a sample preparation based on protein precipitation.

Body fluid and tissue analysis using filter paper sampling support prior to LC-MS/MS: application to fatal overdose with colchicine

Because of the various matrices available for forensic investigations, the development of versatile analytical approaches allowing the simultaneous determination of drugs is challenging. The aim of this work was to assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform allowing the rapid quantification of colchicine in body fluids and tissues collected in the context of a fatal overdose. For this purpose, filter paper was used as a sampling support and was associated with an automated 96-well plate extraction performed by the LC autosampler itself. The developed method features a 7-min total run time including automated filter paper extraction (2 min) and chromatographic separation (5 min). The sample preparation was reduced to a minimum regardless of the matrix analyzed. This platform was fully validated for dried blood spots (DBS) in the toxic concentration range of colchicine. The DBS calibration curve was applied successfully to quantification in all other matrices (body fluids and tissues) except for bile, where an excessive matrix effect was found. The distribution of colchicine for a fatal overdose case was reported as follows: peripheral blood, 29 ng/ml; urine, 94 ng/ml; vitreous humour and cerebrospinal fluid, < 5 ng/ml; pericardial fluid, 14 ng/ml; brain, < 5 pg/mg; heart, 121 pg/mg; kidney, 245 pg/mg; and liver, 143 pg/mg. Although filter paper is usually employed for DBS, we report here the extension of this alternative sampling support to the analysis of other body fluids and tissues. The developed platform represents a rapid and versatile approach for drug determination in multiple forensic media.

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis.

Background Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. Methods 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. Findings 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12. weeks versus 24 weeks (hazard ratio [HR] 1 98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00,0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population. Interpretation While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA,exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population.

Wipe sampling procedure coupled to LC-MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces.

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC-MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm(-2)) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.

Population pharmacokinetic analysis and pharmacogenetics of raltegravir in HIV-positive and healthy individuals.

The objectives of this study were to characterize raltegravir (RAL) population pharmacokinetics in HIV-positive (HIV(+)) and healthy individuals, identify influential factors, and search for new candidate genes involved in UDP glucuronosyltransferase (UGT)-mediated glucuronidation. The pharmacokinetic analysis was performed with NONMEM. Genetic association analysis was performed with PLINK using the relative bioavailability as the phenotype. Simulations were performed to compare once- and twice-daily regimens. A 2-compartment model with first-order absorption adequately described the data. Atazanavir, gender, and bilirubin levels influenced RAL relative bioavailability, which was 30% lower in HIV(+) than in healthy individuals. UGT1A9*3 was the only genetic variant possibly influencing RAL pharmacokinetics. The majority of RAL pharmacokinetic variability remains unexplained by genetic and nongenetic factors. Owing to the very large variability, trough drug levels might be very low under the standard dosing regimen, raising the question of a potential relevance of therapeutic drug monitoring of RAL in some situations.

The differentiation of fibre- and drug type Cannabis seedlings by gas chromatography/mass spectrometry and chemometric tools

Cannabis cultivation in order to produce drugs is forbidden in Switzerland. Thus, law enforcement authorities regularly ask forensic laboratories to determinate cannabis plant's chemotype from seized material in order to ascertain that the plantation is legal or not. As required by the EU official analysis protocol the THC rate of cannabis is measured from the flowers at maturity. When laboratories are confronted to seedlings, they have to lead the plant to maturity, meaning a time consuming and costly procedure. This study investigated the discrimination of fibre type from drug type Cannabis seedlings by analysing the compounds found in their leaves and using chemometrics tools. 11 legal varieties allowed by the Swiss Federal Office for Agriculture and 13 illegal ones were greenhouse grown and analysed using a gas chromatograph interfaced with a mass spectrometer. Compounds that show high discrimination capabilities in the seedlings have been identified and a support vector machines (SVMs) analysis was used to classify the cannabis samples. The overall set of samples shows a classification rate above 99% with false positive rates less than 2%. This model allows then discrimination between fibre and drug type Cannabis at an early stage of growth. Therefore it is not necessary to wait plants' maturity to quantify their amount of THC in order to determine their chemotype. This procedure could be used for the control of legal (fibre type) and illegal (drug type) Cannabis production.

Analysis of fatty acids in ecstasy tablets

Fatty acids are the basis of so-called stearates which are frequently used as lubricants in the production of ecstasy tablets. Being a product added at the initial tablet production step its composition does not change once the compression is performed. The analysis of fatty acids can therefore provide useful information for a drug intelligence purpose. In this context an appropriate analytical method was developed to improve results already obtained by routine analyses. Considering the small quantity of such fatty acids in ecstasy tablets (not, vert, similar3%) the research focussed on their extraction and concentration. Two different procedures were tested: (1) liquid/liquid extraction using dichloromethane followed by derivatisation and (2) in situ transesterification using bortrifluoride. Analyses were performed by GC-MS. The two procedures were optimized and applied to eight ecstasy seizures, in order to choose one of the procedures for its application to a large ecstasy sample set. They were compared by considering the number of peaks detected and sample amount needed, reproducibility and other technical aspects.

A systematic review and meta-analysis of perineural dexamethasone for peripheral nerve blocks.

We systematically reviewed the safety and efficacy of perineural dexamethasone as an adjunct for peripheral nerve blockade in 29 controlled trials of 1695 participants. We grouped trials by the duration of local anaesthetic action (short- or medium- vs long-term). Dexamethasone increased the mean (95% CI) duration of analgesia by 233 (172-295) min when injected with short- or medium-term action local anaesthetics and by 488 (419-557) min when injected with long-term action local anaesthetics, p < 0.00001 for both. However, these results should be interpreted with caution due to the extreme heterogeneity of results, with I2 exceeding 90% for both analyses. Meta-regression did not show an interaction between dose of perineural dexamethasone (4-10 mg) and duration of analgesia (r2 = 0.02, p = 0.54). There were no differences between 4 and 8 mg dexamethasone on subgroup analysis.

Personalized modeling for drug concentration prediction using Support Vector Machine

Building a personalized model to describe the drug concentration inside the human body for each patient is highly important to the clinical practice and demanding to the modeling tools. Instead of using traditional explicit methods, in this paper we propose a machine learning approach to describe the relation between the drug concentration and patients' features. Machine learning has been largely applied to analyze data in various domains, but it is still new to personalized medicine, especially dose individualization. We focus mainly on the prediction of the drug concentrations as well as the analysis of different features' influence. Models are built based on Support Vector Machine and the prediction results are compared with the traditional analytical models.

Androgen dependence of hirsutism, acne, and alopecia in women: retrospective analysis of 228 patients investigated for hyperandrogenism.

Hirsutism, acne, alopecia, and oligo-amenorrhea are clinical expressions of hyperandrogenism, one of the most frequent endocrine disorders in women of reproductive age. Women referred to our endocrine clinics for skin symptoms of hyperandrogenism underwent a laboratory workup to evaluate hormone measurements and received antiandrogen therapy. We retrospectively analyzed the outcome of 228 consecutive patients investigated over 6 years.Patients with hirsutism had higher levels of androstenedione, dehydroepiandrosterone sulfate (DHEAS), and salivary testosterone; lower levels of sex hormone-binding globulin (SHBG); and a higher prevalence of oligo-amenorrhea than patients with alopecia, while patients with acne showed intermediate values. Hirsutism score correlated positively with androstenedione, DHEAS, and salivary testosterone, and correlated negatively with SHBG; salivary testosterone showed the highest correlation coefficient. Total testosterone was not significantly different among patients with hirsutism, alopecia, or acne, and did not significantly correlate with hirsutism score. Hirsutism and oligo-amenorrhea were the most sensitive symptoms of hyperandrogenism, and no androgenic parameter alone allowed us to identify all cases of hyperandrogenism.Patients of central European origin sought consultation with milder hirsutism scores than patients of southern European origin. There was, however, no difference in the clinical-biological correlation between these groups, arguing against differences in skin sensitivity to androgens.Polycystic ovary syndrome, defined as hyperandrogenism (hirsutism or elevated androgens) and oligo-amenorrhea, was diagnosed in 63 patients (27.6%), an underestimate compared with other reports that include systematic ovarian ultrasound studies. Neither pelvic ultrasound, used in a limited number of cases, nor the luteinizing hormone/follicle-stimulating hormone ratio helped to distinguish patients with polycystic ovary syndrome from the other diagnostic groups. These included hyperandrogenism (hirsutism or elevated androgens) and eumenorrhea (101 patients; 44.3%); normal androgens (acne or alopecia and eumenorrhea) (51 patients; 22.4%); isolated low SHBG (7 patients; 3.1%); nonclassical congenital adrenal hyperplasia (4 patients; 1.8% of total, 4.9% of patients undergoing cosyntropin stimulation tests); and ovarian tumor (2 patients; 0.9%).Ethinylestradiol and high-dose cyproterone acetate treatment lowered the hirsutism score to 53.5% of baseline at 1 year, and was also effective in treating acne and alopecia. The clinical benefit is ascribed to the peripheral antiandrogenic effect of cyproterone acetate as well as the hormone-suppressive effect of this combination. Salivary testosterone showed the most marked proportional decrease of all the androgens under treatment. Cost-effectiveness and tolerance of ethinylestradiol and high-dose cyproterone acetate compared well with other antiandrogenic drug therapies for hirsutism. The less potent therapy with spironolactone only, a peripheral antiandrogen without hormone-suppressive effect, was effective in treating isolated alopecia in patients with normal androgens.

Comparative Study of Chemoembolization, Loadable Beads: In vitro Drug Release and Physical Properties of DC Bead and Hepasphere, Loaded with Doxorubicin and Irinotecan

Purpose To characterize in vitro the loadability, physical properties, and release of irinotecan and doxorubicin from two commercially available embolization microspheres. Materials and Methods DC Bead (500-700 μm) and Hepasphere (400-600 μm) microspheres were loaded with either doxorubicin or irinotecan solutions. Drug amount was quantified with spectrophotometry, bead elasticity was measured under compression, and bead size and loading homogeneity were assessed with microscopy image analysis. Drug release was measured over 1-week periods in saline by using a pharmacopeia flow-through method. Results Almost complete drug loading was obtained for both microsphere types and drugs. Doxorubicin-loaded DC Beads maintained their spherical shape throughout the release. In contrast, Hepaspheres showed less homogeneous doxorubicin loading and, after release, some fractured microspheres. Incomplete doxorubicin release was observed in saline over 1 week (27% ± 2 for DC beads and 18% ± 7 for Hepaspheres; P = .013). About 75% of this amount was released within 2.2 hours for both beads. For irinotecan, complete release was obtained for both types of beads, in a sustained manner over 2-3 hours for DC Beads, and in a significantly faster manner as a 7-minute burst for Hepaspheres. Conclusions The two drug-eluting microspheres could be efficiently loaded with both drugs. Incomplete doxorubicin release was attributed to strong drug-bead ionic interactions. Weaker interactions were observed with irinotecan, which led to faster drug release.

In vivo analysis of efavirenz metabolism in individuals with impaired CYP2A6 function.

INTRODUCTION: The antiretroviral drug efavirenz (EFV) is extensively metabolized into three primary metabolites: 8-hydroxy-EFV, 7-hydroxy-EFV and N-glucuronide-EFV. There is a wide interindividual variability in EFV plasma exposure, explained to a great extent by cytochrome P450 2B6 (CYP2B6), the main isoenzyme responsible for EFV metabolism and involved in the major metabolic pathway (8-hydroxylation) and to a lesser extent in 7-hydroxylation. When CYP2B6 function is impaired, the relevance of CYP2A6, the main isoenzyme responsible for 7-hydroxylation may increase. We hypothesize that genetic variability in this gene may contribute to the particularly high, unexplained variability in EFV exposure in individuals with limited CYP2B6 function. METHODS: This study characterized CYP2A6 variation (14 alleles) in individuals (N=169) previously characterized for functional variants in CYP2B6 (18 alleles). Plasma concentrations of EFV and its primary metabolites (8-hydroxy-EFV, 7-hydroxy-EFV and N-glucuronide-EFV) were measured in different genetic backgrounds in vivo. RESULTS: The accessory metabolic pathway CYP2A6 has a critical role in limiting drug accumulation in individuals characterized as CYP2B6 slow metabolizers. CONCLUSION: Dual CYP2B6 and CYP2A6 slow metabolism occurs at significant frequency in various human populations, leading to extremely high EFV exposure.