Infected erythrocyte choline carrier inhibitors: exploring some potentialities inside Plasmodium phospholipid metabolism for eventual resistance acquisition

We have developed a model for designing antimalarial drugs based on interference with an essential metabolism developed by Plasmodium during its intraerythrocytic cycle, phospholipid (PL) metabolism. The most promising drug interference is choline transporter blockage, which provides Plasmodium with a supply of precursor for synthesis of phosphatidylcholine (PC), the major PL of infected erythrocytes. Choline entry is a limiting step in this metabolic pathway and occurs by a facilitated-diffusion system involving an asymmetric carrier operating according to a cyclic model. Choline transport in the erythrocytes is not sodium dependent nor stereospecific as demonstrated using stereoisomers of alpha and beta methylcholine. These last two characteristics along with distinct effects of nitrogen substitution on transport rate demonstrate that choline transport in the infected erythrocyte possesses characteristics quite distinct from that of the nervous system. This indicates a possible discrimination between the antimalarial activity (inhibition of choline transport in the infected erythrocyte) and a possible toxic effect through inhibition of choline entry in synaptosomes. Apart from the de novo pathway of choline, PC can be synthesized by N-methylation from phosphatidylethanolamine (PE). There is a de novo pathway for PE biosynthesis from ethanolamine in infected cells but phosphatidylserine (PS) decarboxylation also occurs. In addition, PE can be directly and abundantly synthesized from serine decarboxylation into ethanolamine, a pathway which is absent from the host. The variety of the pathways that exist for the biosynthesis of one given PL led us to investigate whether an equilibrium can occur between all PL metabolic pathways. Indeed, if alternative (compensative) pathway(s) can operate after blockage of the de novo PC biosynthesis pathway this would indicate a potential mechanism for resistance acquisition. Up until now, there is no evidence of such a compensative process occurring in Plasmodium-infected erythrocytes under physiological conditions. Besides, the discovery of a highly parasite-specific pathway (serine decarboxylation and the presence of PS synthase) constitutes a very attractive and promising target, which could be attacked if resistances are built up against choline analogs. Indeed, potential inhibitions of the serine decarboxylase pathway could be very useful in acting instead of, or in surgery with, choline analogs.

Systemic effects of epidural Methylprednisolone injection on glucose tolerance in diabetic patients.

ABSTRACT: BACKGROUND: Several studies have shown that in diabetic patients, the glycemic profile was disturbed after intra-articular injection of corticosteroids. Little is known about the impact of epidural injection in such patients. The goal of this study was double, at first comparing the glycaemic profile in diabetic patients after a unique injection of 80 mg of acetate methylprednisolone either intra-articular or epidural and secondly to compare the amount of systemic diffusion of the drug after both procedures. METHODS: Seventeen patients were included. Glycemic changes were compared in 9 diabetic patients following intra-articular (4 patients) and epidural injections (5 patients). Epidural injections were performed using the sacral route under fluoroscopic control in patients with lumbar spinal stenosis. Diabetes control had to stable for more than 10 days and the renal function to be preserved. Blood glucose was monitored using a validated continuous measuring device (GMS, Medtronic) the day before and for two days following the injection. Results were expressed in the form of daily glycemic profiles and as by mean, peak and minimal values +/ SD. The urinary excretion of methylprednisolone after the 2 routes of injection was analyzed in 8 patients (4 in each group). Urine samples were cropped one hour before the injections, then 4 times during the first day and 3 times a week for 2 weeks. The measurements included the free and conjugated fraction RESULTS: The glycaemic profile remains unchanged with no significant changes in the group of the 5 diabetic patients receiving epidural injections. On the other end, the average peak and mean values were enhanced up to 3 mmol/l above baseline two days after the infiltration in the groups of the 4 diabetic patients infiltrated intra-articular. The mean urinary excretion of the steroid was about ten times higher in the intra-articular versus epidural group: 7000 ng/ml versus 700 ng/ml. Looking at each individual there were marked differences especially after intra-articular injections. CONCLUSION: This is the first study to show that a single epidural steroid injection of 80 mg depot methylprednisolone had no effect on the glycemic control in diabetic patients. The absence of glycemic control changes correlated well with the very low urinary excretion of the drug after epidural injection. Trial registration NCT01420497.

Stabilized Petrov-Galerkin methods for the convection-diffusion-reaction and the Helmholtz equations

We present two new stabilized high-resolution numerical methods for the convection–diffusion–reaction (CDR) and the Helmholtz equations respectively. The work embarks upon a priori analysis of some consistency recovery procedures for some stabilization methods belonging to the Petrov–Galerkin framework. It was found that the use of some standard practices (e.g. M-Matrices theory) for the design of essentially non-oscillatory numerical methods is not feasible when consistency recovery methods are employed. Hence, with respect to convective stabilization, such recovery methods are not preferred. Next, we present the design of a high-resolution Petrov–Galerkin (HRPG) method for the 1D CDR problem. The problem is studied from a fresh point of view, including practical implications on the formulation of the maximum principle, M-Matrices theory, monotonicity and total variation diminishing (TVD) finite volume schemes. The current method is next in line to earlier methods that may be viewed as an upwinding plus a discontinuity-capturing operator. Finally, some remarks are made on the extension of the HRPG method to multidimensions. Next, we present a new numerical scheme for the Helmholtz equation resulting in quasi-exact solutions. The focus is on the approximation of the solution to the Helmholtz equation in the interior of the domain using compact stencils. Piecewise linear/bilinear polynomial interpolation are considered on a structured mesh/grid. The only a priori requirement is to provide a mesh/grid resolution of at least eight elements per wavelength. No stabilization parameters are involved in the definition of the scheme. The scheme consists of taking the average of the equation stencils obtained by the standard Galerkin finite element method and the classical finite difference method. Dispersion analysis in 1D and 2D illustrate the quasi-exact properties of this scheme. Finally, some remarks are made on the extension of the scheme to unstructured meshes by designing a method within the Petrov–Galerkin framework.

Capsule permeability via polymer and protein ingress/egress.

Static incubation tests, where microcapsules and beads are contacted with polymer and protein solutions, have been developed for the characterization of permselective materials applied for bioartificial organs and drug delivery. A combination of polymer ingress, detected by size-exclusion chromatography, and protein ingress/ egress, assessed by gel electrophoresis, provides information regarding the diffusion kinetics, molar mass cutoff(MMCO) and permeability. This represents an improvement over existing permeability measurements that are based on the diffusion of a single type of solute. Specifically, the permeability of capsules based on alginate, cellulose sulfate, polymethylene-co-guanidine were characterized as a function of membrane thickness. Solid alginate beads were also evaluated. The MMCO of these capsules was estimated to be between 80 and 90 kDa using polymers, and between 116-150 kDa with proteins. Apparently, the globular shape of the proteins (radius of gyration (Rg) of 4.2-4.6 nm) facilitates their passage through the membrane, comparatively to the polysaccharide coil conformation (Rg of 6.5-8.3 nm). An increase of the capsule membrane thickness reduced these values. The MMCO of the beads, which do not have a membrane limiting their permselective properties, was higher, between 110 and 200 kDa with dextrans, and between 150 and 220 kDa with proteins. Therefore, although the permeability estimated with biologically relevant molecules is generally higher due to their lower radius of gyration, both the MMCO of synthetic and natural watersoluble polymers correlate well, and can be used as in vitro metrics for the immune protection ability of microcapsules and microbeads. This article shows, to the authors' knowledge, the first reported concordance between permeability measures based on model natural and biological macromolecules.

Evaluation of the impact of a therapeutic drug monitoring program in a Swiss University Hospital

Background and objective: Therapeutic Drug Monitoring (TDM) has been introduced early 1970 in our hospital (CHUV). It represents nowadays an important routine activity of the Division of Clinical Pharmacology and Toxicology (PCL), and its impact and utility for clinicians required assessment. This study thus evaluated the impact of TDM recommendations in terms of dosage regimen adaptation. Design: A prospective observational study was conducted over 5 weeks. The primary objective was to evaluate the application of our TDM recommendations and to identify potential factors associated to variations in their implementation. The secondary objective was to identify pre-analytical problems linked to the collection and processing of blood samples. Setting: Four representative clinical units at CHUV. Main outcome measure: Clinical data, drug related data (intake, collection and processing) and all information regarding the implementation of clinical recommendations were collected and analyzed by descriptive statistics. Results: A total of 241 blood measurement requests were collected, among which 105 triggered a recommendation. 37% of the recommendations delivered were applied, 25 % partially applied and 34% not applied. In 4% it was not applicable. The factors determinant for implementation were the clinical unit and the mode of transmission of the recommendation (written vs oral). No clear difference between types of drugs could be detected. Pre-analytical problems were not uncommon, mostly related to completion of request forms and delays in blood sampling (equilibration or steady-state not reached). We have identified 6% of inappropriate and unusable drug level measurements that could cause a substantial cost for the hospital. Conclusion: This survey highlighted a better implementation of TDM recommendations in clinical units where this routine is well integrated and understood by the medical staff. Our results emphasize the importance of communication with the nurse or the physician in charge, either to transmit clinical recommendations or to establish consensual therapeutic targets in specific conditions. Development of strong partnerships between clinical pharmacists or pharmacologists and clinical units would be beneficial to improve the impact of this clinical activity.

Docking, virtual high throughput screening and in silico fragment-based drug design.

ABSTRACT The drug discovery process has been profoundly changed recently by the adoption of computational methods helping the design of new drug candidates more rapidly and at lower costs. In silico drug design consists of a collection of tools helping to make rational decisions at the different steps of the drug discovery process, such as the identification of a biomolecular target of therapeutical interest, the selection or the design of new lead compounds and their modification to obtain better affinities, as well as pharmacokinetic and pharmacodynamic properties. Among the different tools available, a particular emphasis is placed in this review on molecular docking, virtual high throughput screening and fragment-based ligand design.

Individual contributions of mutant protease and reverse transcriptase to viral infectivity, replication, and protein maturation of antiretroviral drug-resistant human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease (PR) and reverse transcriptase (RT) inhibitors may display impaired infectivity and replication capacity. The individual contributions of mutated HIV-1 PR and RT to infectivity, replication, RT activity, and protein maturation (herein referred to as "fitness") in recombinant viruses were investigated by separately cloning PR, RT, and PR-RT cassettes from drug-resistant mutant viral isolates into the wild-type NL4-3 background. Both mutant PR and RT contributed to measurable deficits in fitness of viral constructs. In peripheral blood mononuclear cells, replication rates (means +/- standard deviations) of RT recombinants were 72.5% +/- 27.3% and replication rates of PR recombinants were 60.5% +/- 33.6% of the rates of NL4-3. PR mutant deficits were enhanced in CEM T cells, with relative replication rates of PR recombinants decreasing to 15.8% +/- 23.5% of NL4-3 replication rates. Cloning of the cognate RT improved fitness of some PR mutant clones. For a multidrug-resistant virus transmitted through sexual contact, RT constructs displayed a marked infectivity and replication deficit and diminished packaging of Pol proteins (RT content in virions diminished by 56.3% +/- 10.7%, and integrase content diminished by 23.3% +/- 18.4%), a novel mechanism for a decreased-fitness phenotype. Despite the identified impairment of recombinant clones, fitness of two of the three drug-resistant isolates was comparable to that of wild-type, susceptible viruses, suggestive of extensive compensation by genomic regions away from PR and RT. Only limited reversion of mutated positions to wild-type amino acids was observed for the native isolates over 100 viral replication cycles in the absence of drug selective pressure. These data underscore the complex relationship between PR and RT adaptive changes and viral evolution in antiretroviral drug-resistant HIV-1.

Médicaments et grossesse : modifications pharmacocinétiques et place du suivi thérapeutique pharmacologique [Pharmacokinetic Alterations in Pregnancy and Use of Therapeutic Drug Monitoring].

Following the thalidomide tragedy, pharmacological research in pregnant women focused primarily on drug safety for the unborn child and remains only limited regarding the efficacy and safety of treatment for the mother. Significant physiological changes during pregnancy may yet affect the pharmacokinetics of drugs and thus compromise its efficacy and/or safety. Therapeutic drug monitoring (TDM) would maximize the potential effectiveness of treatments, while minimizing the potential risk of toxicity for the mother and the fetus. At present, because of the lack of concentration-response relationship studies in pregnant women, TDM can rely only on individual assessment (based on an effective concentration before pregnancy) and remains reserved only to unexpected situations such as signs of toxicity or unexplained inefficiency.

Suivi de l'adhésion thérapeutique des patients hypertendus ou dyslipi- démiques par les cercles de qualité médecins-pharmaciens [Monitoring drug adherence for hypertensive and dyslipidemic patients in networks of community-based pharmacists and physicians].

Patient adherence is often poor for hypertension and dyslipidaemia. A monitoring of drug adherence might improve these risk factors control, but little is known in ambulatory care. We conducted a randomised controlled study in networks of community-based pharmacists and physicians in the canton of Fribourg to examine whether monitoring drug adherence with an electronic monitor (MEMS) would improve risk factor control among treated, but uncontrolled hypertensive and dyslipidemic patients. The results indicate that MEMS achieve a better blood pressure control and lipid profile, although its implementation requires considerable resources. The study also shows the value of collaboration between physicians and pharmacists in the field of patient adherence to improve ambulatory care of patients with cardiovascular risk factors.

The rapid urinary drug toxicology screen in the emergency room: a useful tool ?

Intoxications are a frequent problem in the ER. In the vast majorityof cases, supportive treatment is sufficient. Severe intoxications withunknown agents are considered an indication for a urinary drug screen,and are recommended by several toxicology centers. However, theirusefulness for patient management remains uncertain.Study objectives: Evaluation of the impact of a urinary drug screen(Biosite Triage TOX Drug Screen) testing 11 substances(acetaminophen, amphetamines, methamphetamines, barbiturates,benzodiazepines, cocaïne, methadone, opioids, phencyclidine,cannabis, tricyclic antidepressants) on initial adult patient managementin the emergency department of a university hospital with ~35.000annual admissions.Methods: Observational retrospective analysis of all tests performedbetween 09/2009 and 09/2010. A test utility was defined as useful if itresulted in the administration of a specific antidote (Flumazenil/Naloxone), the use of a quantitative confirmatory toxicologic test, or achange in patient's disposition.Results: 57 tests were performed. Patient age was 32 ± 11 (SD) years;58% were men; 30% were also intoxicated with alcohol. Two patientsdied (3.5%): the first one of a diphenhydramin overdose, the other of ahypertensive intracerebral hemorrhage believed to be caused cocaineabuse but a negative urine test. Test indications were: 54% firstpsychotic episode; 25% acute respiratory failure; 18% coma; 12%seizure; 11% opioids toxidrome; 7% sympathicomimetic toxidrome; 5%hypotension; 4% ventricular arrhythmia (VT, VF, torsades de pointes)or long QT. 75% of tests were positives for >=1 substance (mean 1.7 ±0.9). 47% of results were unexpected by history. 18% of resultsinfluenced patient management: 7% had a negative test that confirmedthe diagnosis of endogenous psychosis in a first psychotic episode, andallowed transfer to psychiatry; 5% received flumazenil/naloxone;2% had an acetaminophen blood level after a positive screen; finally,4% had an unexpected methadone abuse that required prolongationof hospital stay.Conclusions: A rapid urinary toxicologic screen was seldom used inour emergency department, and its impact on patient managementwas marginal: only one in 6 tests influenced treatment decisions.

A geometric mechanism of diffusion: Rigorous verification in a priori unstable Hamiltonian systems

In this paper we consider a representative a priori unstable Hamiltonian system with 2+1/2 degrees of freedom, to which we apply the geometric mechanism for diffusion introduced in the paper Delshams et al., Mem.Amer.Math. Soc. 2006, and generalized in Delshams and Huguet, Nonlinearity 2009, and provide explicit, concrete and easily verifiable conditions for the existence of diffusing orbits. The simplification of the hypotheses allows us to perform explicitly the computations along the proof, which contribute to present in an easily understandable way the geometric mechanism of diffusion. In particular, we fully describe the construction of the scattering map and the combination of two types of dynamics on a normally hyperbolic invariant manifold.