Convenient synthesis of heterobifunctional poly(ethylene glycol) suitable for the functionalization of iron oxide nanoparticles for biomedical applications.

A straightforward route is proposed for the multi-gram scale synthesis of heterobifunctional poly(ethylene glycol) (PEG) oligomers containing combination of triethyloxysilane extremity for surface modification of metal oxides and amino or azido active end groups for further functionalization. The suitability of these PEG derivatives to be conjugated to nanomaterials was shown by pegylation of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (NPs), followed by functionalization with small peptide ligands for biomedical applications.

Dipeptidyl peptidase IV and its inhibitors: therapeutics for type 2 diabetes and what else?

The proline-specific dipeptidyl aminopeptidase IV (DPP IV, DPP-4, CD26), widely expressed in mammalians, releases X-Pro/Ala dipeptides from the N-terminus of peptides. DPP IV is responsible of the degradation of the incretin peptide hormones regulating blood glucose levels. Several families of DPP IV inhibitors have been synthesized and evaluated. Their positive effects on the degradation of the incretins and the control of blood glucose levels have been demonstrated in biological models and in clinical trials. Presently, several DPP IV inhibitors, the "gliptins", are approved for type 2 diabetes or are under clinical evaluation. However, the gliptins may also be of therapeutic interest for other diseases beyond the inhibition of incretin degradation. In this Perspective, the biological functions and potential substrates of DPP IV enzymes are reviewed and the characteristics of the DPP IV inhibitors are discussed in view of type 2 diabetes and further therapeutic interest.

Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

Anti-cancer activity of 5-O-alkyl 1,4-imino-1,4-dideoxyribitols.

New derivatives of 1,4-dideoxy-1,4-imino-D-ribitol have been prepared and evaluated for their cytotoxicity on solid and haematological malignancies. 1,4-Dideoxy-5-O-[(9Z)-octadec-9-en-1-yl]-1,4-imino-D-ribitol (13, IC(50) ∼2 μM) and its C(18)-analogues (IC(50) <10 μM) are cytotoxic toward SKBR3 (breast cancer) cells. 13 also inhibits (IC(50) ∼8 μM) growth of JURKAT cells.

PPARs in diseases: control mechanisms of inflammation.

The three isotypes of peroxisome proliferator-activated receptors (PPARs), PPARalpha, beta/delta and gamma, are ligand-inducible transcription factors that belong to the nuclear hormone receptor family. PPARs are implicated in the control of inflammatory responses and in energy homeostasis and thus, can be defined as metabolic and anti-inflammatory transcription factors. They exert their anti-inflammatory effects by inhibiting the induction of pro-inflammatory cytokines, adhesion molecules and extracellular matrix proteins or by stimulating the production of anti-inflammatory molecules. Furthermore, PPARs modulate the proliferation, differentiation and survival of immune cells including macrophages, B cells and T cells. This review discusses the molecular mechanisms by which PPARs and their ligands modulate the inflammatory response. In addition, it presents recent developments implicating PPAR specific ligands in potential treatments of inflammation-related diseases, such as atherosclerosis, inflammatory bowel diseases, Parkinson's and Alzheimer's diseases.

Synthesis and in vitro evaluation of a novel radioligand for αvβ3 integrin receptor imaging: [(18)F]FPPA-c(RGDfK).

The development of RGD-based antagonist of αvβ3 integrin receptor has enhanced the interest in PET probes to image this receptor for the early detection of cancer, to monitor the disease progression and the response to therapy. In this work, a novel prosthetic group (N-(4-fluorophenyl)pent-4-ynamide or FPPA) for the (18)F-labeling of an αvβ3 selective RGD-peptide was successfully prepared. [(18)F]FPPA was obtained in three steps with a radiochemical yield of 44% (decay corrected). Conjugation to c(RGDfK(N3)) by the Cu(II) catalyzed Huisgen azido alkyne cycloaddition provided the [(18)F]FPPA-c(RGDfK) with a radiochemical yield of 29% (decay corrected), in an overall synthesis time of 140min.

Smoked cannabis and doping control: looking for the wrong target analyte?

Introduction: Since 2004, cannabis is prohibited by the World Anti-Doping Agency (WADA) for all sports in competition. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In most cases, the athletes plausibly claim to have consumed cannabis several days or even weeks before competition and only for recreational purposes not related to competition. In doping analysis, the target analyte in urine samples is 11-nor-delta-9-tetrahydrocannabinol- 9-carboxylic acid (THC-COOH), the reporting threshold for laboratories is 15 ng/mL. However, the wide detection window of this long-term THC metabolite in urine does not allow a conclusion concerning the time of consumption or the impact on the physical performance. Aim: The purpose of the present pharmacokinetic study on volunteers was to evaluate target analytes with shorter urinary excretion time. Subsequently, urines from athletes tested positive for cannabis should be reanalyzed including these analytes. Methods: In an one-session clinical trial (approved by IRB, Swissmedic, and Federal Office of Public Health), 12 healthy, male volunteers (age 26 ± 3 yrs, BMI 24 ± 2 kg/m2) with cannabis experience (> once/month) smoked a Cannabis cigarette standardized to 70 mg THC/cigarette (Bedrobinol® 7%, Dutch Office for Medicinal Cannabis) following a paced-puffing procedure. Plasma and urine was collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-THC (THC-OH), and THC-COOH were determined after enzymatic hydrolyzation followed by SPE and GC/MS-SIM. The limit of quantitation (LOQ) for all analytes was 0.1 ng/mL. Visual analog scales (VAS) and vital functions were used for monitoring psychological and somatic side-effects at every timepoint of specimen collection (up to 480 min). Results: Eight puffs delivered a mean THC dose of 45 mg. Mean plasma levels of total THC, THC-OH and THC-COOH were measured in the range of 0.1-20.9, 0.1-1.8, and 1.8-7.5 ng/mL, respectively. Peak concentrations were observed at 5, 10, and 90 min. Mean urine levels were measured in the range of 0.1-0.7, 0.10-6.2, and 0.1-13.4 ng/mL, respectively. The detection windows were 2-8, 2-96, and 2-120 h. No or only mild effects were observed, such as dry mouth, sedation, and tachycardia. Besides high to very high THC-COOH levels (0-978 ng/mL), THC (0.1-24 ng/mL) and THC-OH (1-234 ng/mL) were found in 90 and 96% of the cannabis-positive urines from athletes. Conclusion: Instead of or in addition to THC-COOH, the pharmacologically active THC and THC-OH should be the target analytes for doping urine analysis. This would allow the estimation of more recent Cannabis consumption, probably influencing performance during competition. Keywords: cannabis, doping, clinical trial, plasma and urine levels, athlete's samples

Fenofibrate: a new treatment for diabetic retinopathy. Molecular mechanisms and future perspectives.

Despite improving standards of care, people with diabetes remain at risk of development and progression of diabetic retinopathy (DR) and visual impairment. Identifying novel therapeutic approaches, preferably targeting more than one pathogenic pathway in DR, and at an earlier stage of disease, is attractive. There is now consistent evidence from two major trials, the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study and the Action to Control Cardiovascular Risk in Diabetes Eye (ACCORD-Eye) study, totalling 11,388 people with type 2 diabetes (5,701 treated with fenofibrate) that fenofibrate reduces the risk of development and progression of DR. Therefore, fenofibrate may be considered a preventive strategy for patients without DR or early intervention strategy for those with mild DR. A number of putative therapeutic mechanisms for fenofibrate, both dependent and independent of lipids, have been proposed. A deeper understanding of the mode of action of fenofibrate will further help to define how best to use fenofibrate clinically as an adjunct to current management of DR.

Temozolomide: a milestone in neuro-oncology and beyond?

Temozolomide (Temodal, Temodar), an imidazol derivative, is a second-generation alkylating agent. The orally available prodrug with the capacity of crossing the blood-brain barrier received accelerated US FDA approval in 1999. Three pivotal Phase II trials showed modest activity in the treatment of recurrent anaplastic astrocytoma glioblastoma. In 2005, the FDA and the European Agency for the Evaluation of Medicinal Products approved temozolomide for use in newly diagnosed glioblastoma, in conjunction with radiotherapy, based on an European Organisation for Research and Treatment of Cancer/National Cancer Institute of Canada Phase III trial. The adverse events associated with temozolomide are mild-to-moderate and generally predictable; the most serious are noncumulative and reversible myelosuppression and, in particular, thrombocytopenia, which occurs in less than 5% of patients. Continuous temozolomide administration is associated with profound CD4-selective lymphocytopenia. Molecular studies have suggested that the benefit of temozolomide chemotherapy is restricted to patients whose tumors have a methylated methylguanine methyltransferase gene promotor and are thus unable to repair some of the chemotherapy-induced DNA damage. Temozolomide is under investigation for other disease entities, in particular lower-grade glioma, brain metastases and melanoma.

Hemoglobin enhances the biological activity of synthetic and natural bacterial (endotoxic) virulence factors: a general principle.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Development of a Selective Peptide Macrocycle Inhibitor of Coagulation Factor XII toward the Generation of a Safe Antithrombotic Therapy.

Inhibition of coagulation factor XII (FXII) activity represents an attractive approach for the treatment and prevention of thrombotic diseases. The few existing FXII inhibitors suffer from low selectivity. Using phage display combined to rational design, we developed a potent inhibitor of FXII with more than 100-fold selectivity over related proteases. The highly selective peptide macrocycle is a promising candidate for the control of FXII activity in antithrombotic therapy and a valuable tool in hematology research.

Database construction and Peptide identification strategies for proteogenomic studies on sequenced genomes.

Since the advent of high-throughput DNA sequencing technologies, the ever-increasing rate at which genomes have been published has generated new challenges notably at the level of genome annotation. Even if gene predictors and annotation softwares are more and more efficient, the ultimate validation is still in the observation of predicted gene product( s). Mass-spectrometry based proteomics provides the necessary high throughput technology to show evidences of protein presence and, from the identified sequences, confirmation or invalidation of predicted annotations. We review here different strategies used to perform a MS-based proteogenomics experiment with a bottom-up approach. We start from the strengths and weaknesses of the different database construction strategies, based on different genomic information (whole genome, ORF, cDNA, EST or RNA-Seq data), which are then used for matching mass spectra to peptides and proteins. We also review the important points to be considered for a correct statistical assessment of the peptide identifications. Finally, we provide references for tools used to map and visualize the peptide identifications back to the original genomic information.

First nationwide study on driving under the influence of drugs in Switzerland.

In Switzerland, a two-tier system based on impairment by any psychoactive substances which affect the capacity to drive safely and zero tolerance for certain illicit drugs came into force on 1 January 2005. According to the new legislation, the offender is sanctioned if Delta(9)-tetrahydrocannabinol THC is >or=1.5ng/ml or amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), cocaine, free morphine are >or=15ng/ml in whole blood (confidence interval+/-30%). For all other psychoactive substances, impairment must be proven in applying the so-called "three pillars expertise". At the same time the legal blood alcohol concentration (BAC) limit for driving was lowered from 0.80 to 0.50g/kg. The purpose of this study was to analyze the prevalence of drugs in the first year after the introduction of the revision of the Swiss Traffic Law in the population of drivers suspected of driving under the influence of drugs (DUID). A database was developed to collect the data from all DUID cases submitted by the police or the Justice to the eight Swiss authorized laboratories between January and December 2005. Data collected were anonymous and included the age, gender, date and time of the event, the type of vehicle, the circumstances, the sampling time and the results of all the performed toxicological analyses. The focus was explicitly on DUID; cases of drivers who were suspected to be under the influence of ethanol only were not considered. The final study population included 4794 DUID offenders (4243 males, 543 females). The mean age of all drivers was 31+/-12 years (range 14-92 years). One or more psychoactive drugs were detected in 89% of all analyzed blood samples. In 11% (N=530) of the samples, neither alcohol nor drugs were present. The most frequently encountered drugs in whole blood were cannabinoids (48% of total number of cases), ethanol (35%), cocaine (25%), opiates (10%), amphetamines (7%), benzodiazepines (6%) and methadone (5%). Other medicinal drugs such as antidepressants and benzodiazepine-like were detected less frequently. Poly-drug use was prevalent but it may be underestimated because the laboratories do not always analyze all drugs in a blood sample. This first Swiss study points out that DUID is a serious problem on the roads in Switzerland. Further investigations will show if this situation has changed in the following years.

Rational design of indoleamine 2,3-dioxygenase inhibitors.

Indoleamine 2,3-dioxygenase (IDO) is an important therapeutic target for the treatment of diseases such as cancer that involve pathological immune escape. We have used the evolutionary docking algorithm EADock to design new inhibitors of this enzyme. First, we investigated the modes of binding of all known IDO inhibitors. On the basis of the observed docked conformations, we developed a pharmacophore model, which was then used to devise new compounds to be tested for IDO inhibition. We also used a fragment-based approach to design and to optimize small organic molecule inhibitors. Both approaches yielded several new low-molecular weight inhibitor scaffolds, the most active being of nanomolar potency in an enzymatic assay. Cellular assays confirmed the potential biological relevance of four different scaffolds.