Iptakalim: A Potential Antipsychotic Drug with Novel Mechanisms?

Iptakalim is a novel putative adenosine triphosphate (ATP)-sensitive potassium (KATP) channel opener. In the brain, iptakalim is thought to act on the neuronal and astrocytic plasma membrane and/or mitochondrial KATP channels. Because iptakalim demonstrates an action on the regulation of dopamine and glutamate release in the forebrain regions, we examined its potential antipsychotic efficacy in several preclinical tests. First, we show that iptakalim is effective in reducing amphetamine- and phencyclidine-induced hyperlocomotion as well as selectively disrupting conditioned avoidance responding. Next, we show that combined iptakalim and amphetamine treatment produces a reduction on prepulse inhibition of acoustic startle and this combined drug effect is also found with haloperidol, but not with clozapine. Finally, we show that iptakalim and clozapine preferentially increase c-Fos expression in the medial prefrontal cortex, nucleus accumbens and lateral septal nucleus, whereas haloperidol induces a greater increase in the nucleus accumbens, the dorsolateral striatum and lateral septal nucleus. Collectively, our findings indicate that iptakalim is likely to be a potential antipsychotic drug with distinct mechanisms of action. This study also suggests that neuronal and astrocytic plasma membrane and/or mitochondrial KATP channels may be a novel target that deserves attention for antipsychotic drug development. Future research using other sensitive tests is needed to confirm this property of iptakalim.

Emtricitabine-Tenofovir Concentrations and Pre-Exposure Prophylaxis Efficacy in Men Who Have Sex with Men

Drug concentrations associated with protection from HIV-1 acquisition have not been determined. We evaluated drug concentrations among men who have sex with men in a substudy of the iPrEx trial (1). In this randomized placebo-controlled trial, daily oral doses of emtricitabine/tenofovir disoproxil fumarate were used as pre-exposure prophylaxis (PrEP) in men who have sex with men. Drug was detected less frequently in blood plasma and in viable cryopreserved peripheral blood mononuclear cells (PBMCs) in HIV-infected cases at the visit when HIV was first discovered compared with controls at the matched time point of the study (8% versus 44%; P < 0.001) and in the 90 days before that visit (11% versus 51%; P < 0.001). An intracellular concentration of the active form of tenofovir, tenofovir-diphosphate (TFV-DP), of 16 fmol per million PBMCs was associated with a 90% reduction in HIV acquisition relative to the placebo arm. Directly observed dosing in a separate study, the STRAND trial, yielded TFV-DP concentrations that, when analyzed according to the iPrEx model, corresponded to an HIV-1 risk reduction of 76% for two doses per week, 96% for four doses per week, and 99% for seven doses per week. Pro-phylactic benefits were observed over a range of doses and drug concentrations, suggesting ways to optimize PrEP regimens for this population.

Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

Background: Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance. Methods/Results: We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in Sao Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naive individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples. Conclusion: The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3-5 x less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.

US Food and Drug Administration approval of generic versions of complex biologics: implications for the practicing physician using low molecular weight heparins

Low-molecular-weight heparins (LMWHs) have shown equivalent or superior efficacy and safety to unfractionated heparin as antithrombotic therapy for patients with acute coronary syndromes. Each approved LMWH is a pleotropic biological agent with a unique chemical, biochemical, biophysical and biological profile and displays different pharmacodynamic and pharmacokinetic profiles. As a result, LMWHs are neither equipotent in preclinical assays nor equivalent in terms of their clinical efficacy and safety. Previously, the US Food and Drug Administration (FDA) cautioned against using various LMWHs interchangeably, however recently, the FDA approved generic versions of LMWH that have not been tested in large clinical trials. This paper highlights the bio-chemical and pharmacological differences between the LMWH preparations that may result in different clinical outcomes, and also reviews the implications and challenges physicians face when generic versions of the original/innovator agents are approved for clinical use.

Solid Dispersion of Ursolic Acid in Gelucire 50/13: a Strategy to Enhance Drug Release and Trypanocidal Activity

Solid dispersions (SDs) are an approach to increasing the water solubility and bioavailability of lipophilic drugs such as ursolic acid (UA), a triterpenoid with trypanocidal activity. In this work, Gelucire 50/13, a surfactant compound with permeability-enhancing properties, and silicon dioxide, a drying adjuvant, were employed to produce SDs with UA. SDs and physical mixtures (PMs) in different drug/carrier ratios were characterized and compared using differential scanning calorimetry, hot stage microscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), particle size, water solubility values, and dissolution profiles. Moreover, LLC-MK2 fibroblast cytotoxicity and trypanocidal activity evaluation were performed to determine the potential of SD as a strategy to improve UA efficacy against Chagas disease. The results demonstrated the conversion of UA from the crystalline to the amorphous state through XRD. FTIR experiments provided evidence of intermolecular interactions among the drug and carriers through carbonyl peak broadening in the SDs. These findings helped explain the enhancement of water solubility from 75.98 mu g/mL in PMs to 293.43 mu g/mL in SDs and the faster drug release into aqueous media compared with pure UA or PMs, which was maintained after 6 months at room temperature. Importantly, improved SD dissolution was accompanied by higher UA activity against trypomastigote forms of Trypanosoma cruzi, but not against mammalian fibroblasts, enhancing the potential of UA for Chagas disease treatment.

Assessment of Preemptive Analgesia Efficacy in Surgical Extraction of Third Molars

Background and objectives: Literature on preemptive analgesia is controversial. Reliability of results and difficult reproducibility of research contribute for non-elucidation of the subject. The aim of this study is to test the efficacy of oral ketoprofen (150 mg) preemptively administrated two days before third molar surgery, compared with postoperative administration in the same patient. Methods: Thirteen patients underwent surgical removal of bilateral third molar in two separate procedures. In a random and double blind procedure, oral ketoprofen 150 mg was administered every 12 hours two days before surgery and, after the procedure, the same drug was administered for three days. On the other side, a control (placebo) was used orally every 12 hours two days before surgery and, after the procedure, ketoprofen 150 mg was administered every 12 hours for three days. Postoperative pain was assessed by visual analogue scale, nominal scale, and amount of rescue analgesics consumed. Results: There was no statistically significant difference in postoperative pain between the preemptive treatment and control. Conclusion: In this experimental model, preemptive analgesia was not effective in reducing postoperative pain in surgical extraction of third molar compared with the postoperative administration of the same drug.

Co-nanoencapsulation of magnetic nanoparticles and selol for breast tumor treatment: in vitro evaluation of cytotoxicity and magnetohyperthermia efficacy

Antitumor activities have been described in selol, a hydrophobic mixture of molecules containing selenium in their structure, and also in maghemite magnetic nanoparticles (MNPs). Both selol and MNPs were co-encapsulated within poly(lactic-co-glycolic acid) (PLGA) nanocapsules for therapeutic purposes. The PLGA-nanocapsules loaded with MNPs and selol were labeled MSE-NC and characterized by transmission and scanning electron microscopy, electrophoretic mobility, photon correlation spectroscopy, presenting a monodisperse profile, and positive charge. The antitumor effect of MSE-NC was evaluated using normal (MCF-10A) and neoplastic (4T1 and MCF-7) breast cell lines. Nanocapsules containing only MNPs or selol were used as control. MTT assay showed that the cytotoxicity induced by MSE-NC was dose and time dependent. Normal cells were less affected than tumor cells. Cell death occurred mainly by apoptosis. Further exposure of MSE-NC treated neoplastic breast cells to an alternating magnetic field increased the antitumor effect of MSE-NC. It was concluded that selol-loaded magnetic PLGA-nanocapsules (MSE-NC) represent an effective magnetic material platform to promote magnetohyperthermia and thus a potential system for antitumor therapy.

Structural Investigation of Anti-Trypanosoma cruzi 2-Iminothiazolidin-4-ones Allows the Identification of Agents with Efficacy in Infected Mice

We modified the thiazolidinic ring at positions N3, C4, and C5, yielding compounds 6-24. Compounds with a phenyl at position N3, 15-19, 22-24, exhibited better inhibitory properties for cruzain and against the parasite than 2-iminothiazolidin-4-one S. We were able to identify one high-efficacy trypanocidal compound, 2-minothiazolidin-4-one 18, which inhibited the activity of cruzain and the proliferation of epirnastigotes and was cidal for trypomastigotes but was not toxic for splenocytes. Having located some of the structural determinants of the trypanocidal properties, we subsequently wished to determine if the exchange of the thiazolidine for a thiazole ring leaves the functional properties unaffected. We therefore tested thiazoles 26-45 and observed that they did not inhibit cruzain, but they exhibited trypanocidal effects. Parasite development was severely impaired when treated with 18, thus reinforcing the notion that this class of heterocycles can lead to useful cidal agents for Chagas disease.

In vivo study of laser irradiation of fractionated drug administration based mechanism for effective photodynamic therapy in rat liver

Up-regulation of stress-activated proteins in cancer cells plays a protective role against photodynamic induced apoptosis. Post photodynamic therapy extracted normal rat liver tissue usually shows a fraction of surviving cells, the photodynamic resistant cells, residing in the necrotic region. To treat these photo-dynamic resistant cells a technique has been proposed based on fractionated drug administration of diluted photosensitizer, keeping the net concentration (5 mg/kg) constant, and subsequently varying drug light interval (DLI). Flourescence measurements were made for the presence of photosensitizer in a tissue. For qualitative analysis both histological and morphological studies were made. Although preliminary aim of this approach was not achieved but there were some interesting observation made i.e. for higher dilution of photosensitizer there was a sharp boundary between necrotic and normal portion of tissue. An increase in the absorption coefficient (alpha) from 2.7 -> 2.9 was observed as photosensitizer was diluted while the corresponding threshold dose (D (th)) persistently decreases from (0.10 -> 0.02) J/cm(2) when irradiated with a 635 nm laser fluence of 150 J/cm(2).

Optimization of anti-Trypanosoma cruzi oxadiazoles leads to identification of compounds with efficacy in infected mice

We recently showed that oxadiazoles have anti-Trypanosoma cruzi activity at micromolar concentrations. These compounds are easy to synthesize and show a number of clear and interpretable structure-activity relationships (SAR), features that make them attractive to pursue potency enhancement. We present here the structural design, synthesis, and anti-T. cruzi evaluation of new oxadiazoles denoted 5a-h and 6a-h. The design of these compounds was based on a previous model of computational docking of oxadiazoles on the T. cruzi protease cruzain. We tested the ability of these compounds to inhibit catalytic activity of cruzain, but we found no correlation between the enzyme inhibition and the antiparasitic activity of the compounds. However, we found reliable SAR data when we tested these compounds against the whole parasite. While none of these oxadiazoles showed toxicity for mammalian cells, oxadiazoles 6c (fluorine), 6d (chlorine), and 6e (bromine) reduced epimastigote proliferation and were cidal for trypomastigotes of T. cruzi Y strain. Oxadiazoles 6c and 6d have IC50 of 9.5 +/- 2.8 and 3.5 +/- 1.8 mu M for trypomastigotes, while Benznidazole, which is the currently used drug for Chagas disease treatment, showed an IC50 of 11.3 +/- 2.8 mu M. Compounds 6c and 6d impair trypomastigote development and invasion in macrophages, and also induce ultrastructural alterations in trypomastigotes. Finally, compound 6d given orally at 50 mg/kg substantially reduces the parasitemia in T. cruzi-infected BALB/c mice. Our drug design resulted in potency enhancement of oxadiazoles as anti-Chagas disease agents, and culminated with the identification of oxadiazole 6d, a trypanosomicidal compound in an animal model of infection. (C) 2012 Elsevier Ltd. All rights reserved.

Anticancer drug screening from images of zebrafish embryogenesis

During the previous 10 years, global R&D expenditure in the pharmaceuticals and biotechnology sector has steadily increased, without a corresponding increase in output of new medicines. To address this situation, the biopharmaceutical industry's greatest need is to predict the failures at the earliest possible stage of the drug development process. A major key to reducing failures in drug screenings is the development and use of preclinical models that are more predictive of efficacy and safety in clinical trials. Further, relevant animal models are needed to allow a wider testing of novel hypotheses. Key to this is the developing, refining, and validating of complex animal models that directly link therapeutic targets to the phenotype of disease, allowing earlier prediction of human response to medicines and identification of safety biomarkers. Morehover, well-designed animal studies are essential to bridge the gap between test in cell cultures and people. Zebrafish is emerging, complementary to other models, as a powerful system for cancer studies and drugs discovery. We aim to investigate this research area designing a new preclinical cancer model based on the in vivo imaging of zebrafish embryogenesis. Technological advances in imaging have made it feasible to acquire nondestructive in vivo images of fluorescently labeled structures, such as cell nuclei and membranes, throughout early Zebrafishsh embryogenesis. This In vivo image-based investigation provides measurements for a large number of features at cellular level and events including nuclei movements, cells counting, and mitosis detection, thereby enabling the estimation of more significant parameters such as proliferation rate, highly relevant for investigating anticancer drug effects. In this work, we designed a standardized procedure for accessing drug activity at the cellular level in live zebrafish embryos. The procedure includes methodologies and tools that combine imaging and fully automated measurements of embryonic cell proliferation rate. We achieved proliferation rate estimation through the automatic classification and density measurement of epithelial enveloping layer and deep layer cells. Automatic embryonic cells classification provides the bases to measure the variability of relevant parameters, such as cell density, in different classes of cells and is finalized to the estimation of efficacy and selectivity of anticancer drugs. Through these methodologies we were able to evaluate and to measure in vivo the therapeutic potential and overall toxicity of Dbait and Irinotecan anticancer molecules. Results achieved on these anticancer molecules are presented and discussed; furthermore, extensive accuracy measurements are provided to investigate the robustness of the proposed procedure. Altogether, these observations indicate that zebrafish embryo can be a useful and cost-effective alternative to some mammalian models for the preclinical test of anticancer drugs and it might also provides, in the near future, opportunities to accelerate the process of drug discovery.

Development of original analytical methods for the therapeutic drug monitoring of CNS druges: Antipsychotics, Antidepressants and Anxiolytics-hypnotics

Great strides have been made in the last few years in the pharmacological treatment of neuropsychiatric disorders, with the introduction into the therapy of several new and more efficient agents, which have improved the quality of life of many patients. Despite these advances, a large percentage of patients is still considered “non-responder” to the therapy, not drawing any benefits from it. Moreover, these patients have a peculiar therapeutic profile, due to the very frequent application of polypharmacy, attempting to obtain satisfactory remission of the multiple aspects of psychiatric syndromes. Therapy is heavily individualised and switching from one therapeutic agent to another is quite frequent. One of the main problems of this situation is the possibility of unwanted or unexpected pharmacological interactions, which can occur both during polypharmacy and during switching. Simultaneous administration of psychiatric drugs can easily lead to interactions if one of the administered compounds influences the metabolism of the others. Impaired CYP450 function due to inhibition of the enzyme is frequent. Other metabolic pathways, such as glucuronidation, can also be influenced. The Therapeutic Drug Monitoring (TDM) of psychotropic drugs is an important tool for treatment personalisation and optimisation. It deals with the determination of parent drugs and metabolites plasma levels, in order to monitor them over time and to compare these findings with clinical data. This allows establishing chemical-clinical correlations (such as those between administered dose and therapeutic and side effects), which are essential to obtain the maximum therapeutic efficacy, while minimising side and toxic effects. It is evident the importance of developing sensitive and selective analytical methods for the determination of the administered drugs and their main metabolites, in order to obtain reliable data that can correctly support clinical decisions. During the three years of Ph.D. program, some analytical methods based on HPLC have been developed, validated and successfully applied to the TDM of psychiatric patients undergoing treatment with drugs belonging to following classes: antipsychotics, antidepressants and anxiolytic-hypnotics. The biological matrices which have been processed were: blood, plasma, serum, saliva, urine, hair and rat brain. Among antipsychotics, both atypical and classical agents have been considered, such as haloperidol, chlorpromazine, clotiapine, loxapine, risperidone (and 9-hydroxyrisperidone), clozapine (as well as N-desmethylclozapine and clozapine N-oxide) and quetiapine. While the need for an accurate TDM of schizophrenic patients is being increasingly recognized by psychiatrists, only in the last few years the same attention is being paid to the TDM of depressed patients. This is leading to the acknowledgment that depression pharmacotherapy can greatly benefit from the accurate application of TDM. For this reason, the research activity has also been focused on first and second-generation antidepressant agents, like triciclic antidepressants, trazodone and m-chlorophenylpiperazine (m-cpp), paroxetine and its three main metabolites, venlafaxine and its active metabolite, and the most recent antidepressant introduced into the market, duloxetine. Among anxiolytics-hypnotics, benzodiazepines are very often involved in the pharmacotherapy of depression for the relief of anxious components; for this reason, it is useful to monitor these drugs, especially in cases of polypharmacy. The results obtained during these three years of Ph.D. program are reliable and the developed HPLC methods are suitable for the qualitative and quantitative determination of CNS drugs in biological fluids for TDM purposes.

Gamma-secretase modulators and inhibitors in Alzheimer's disease: influence of genetic background on efficacy and mechanism of action

According to the amyloid hypothesis, Alzheimer’s disease (AD) is caused by aberrant production or clearance of the amyloid-β (Aβ) peptides, and in particular of the longer more aggregation-prone Aβ42. The Aβ peptides are generated through successive proteolytic cleavage of the amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE) and γ-secretase. γ-secretase produces Aβ peptides with variable C-termini ranging from Aβ34 to Aβ48, presumably by sequential trimming of longer into shorter peptides. γ-secretase is a multiprotein complex consisting of at least four different proteins and the presenilin proteins (PS1 or PS2) contain the catalytic center of the complex. In 2001 several non-steroidal anti-inflammatory drugs were identified as the founding members of a new class of γ-secretase modulators (GSMs) that can selectively reduce production of Aβ42. Concomitantly, these GSMs increase Aβ38 production indicating closely coordinated generation of Aβ42 and Aβ38 and a potential precursor-product relationship between these peptides. GSMs seem to exert their activity by direct modulation of γ-secretase. Support for this hypothesis is drawn from the finding that some PS mutations associated with early-onset familial AD (FAD) can modulate the cellular response to GSMs and to γ-secretase inhibitors (GSIs), which inhibit production of all Aβ peptides and are known to directly interact with PS. A particularly interesting FAD PS mutation is PS1-ΔExon9, a complex deletion mutant that blocks endoproteolysis of PS1 and renders cells completely non-responsive to GSMs. Studies presented in this thesis show that the diminished response of PS1-ΔExon9 to GSMs is mainly caused by its lack of endoproteolytic cleavage. Furthermore, we were able to demonstrate that a reduced response to GSMs and GSIs is not limited to PS1-ΔExon9 but is a common effect of aggressive FAD-associated PS1 mutations. Surprisingly, we also found that while the Aβ42 response to GSMs is almost completely abolished by these PS1 mutations, the accompanying Aβ38 increase was indistinguishable to wild-type PS1. Finally, the reduced response to GSIs was confirmed in a mouse model with transgenic expression of an aggressive FAD-associated PS1 mutation as a highly potent GSI failed to reduce Aβ42 levels in brain of these mice. Taken together, our findings provide clear evidence for independent generation of Aβ42 and Aβ38 peptides, and argue that the sequential cleavage model might be an oversimplification of the molecular mechanism of γ-secretase. Most importantly, our results highlight the significance of genetic background in drug discovery efforts aimed at γ-secretase, and indicate that the use of cellular models with transgenic expression of FAD-associated PS mutations might confound studies of the potency and efficacy of GSMs and GSIs. Therefore, such models should be strictly avoided in the ongoing preclinical development of these promising and potentially disease-modifying therapeutics for AD.

Therapeutisches drug monitoring bei antidepressiver Pharmakotherapie

Therapeutisches Drug Monitoring (TDM) umfasst die Messung von Medikamentenspiegeln im Blut und stellt die Ergebnisse in Zusammenhang mit dem klinischen Erscheinungsbild der Patienten. Dabei wird angenommen, dass die Konzentrationen im Blut besser mit der Wirkung korrelieren als die Dosis. Dies gilt auch für Antidepressiva. Voraussetzung für eine Therapiesteuerung durch TDM ist die Verfügbarkeit valider Messmethoden im Labor und die korrekte Anwendung des Verfahrens in der Klinik. Ziel dieser Arbeit war es, den Einsatz von TDM für die Depressionsbehandlung zu analysieren und zu verbessern. Im ersten Schritt wurde für das neu zugelassene Antidepressivum Duloxetin eine hochleistungsflüssig-chromatographische (HPLC) Methode mit Säulenschaltung und spektrophotometrischer Detektion etabliert und an Patienten für TDM angewandt. Durch Analyse von 280 Patientenproben wurde herausgefunden, dass Duloxetin-Konzentrationen von 60 bis 120 ng/ml mit gutem klinischen Ansprechen und einem geringen Risiko für Nebenwirkungen einhergingen. Bezüglich seines Interaktionspotentials erwies sich Duloxetin im Vergleich zu anderen Antidepressiva als schwacher Inhibitor des Cytochrom P450 (CYP) Isoenzyms 2D6. Es gab keinen Hinweis auf eine klinische Relevanz. Im zweiten Schritt sollte eine Methode entwickelt werden, mit der möglichst viele unterschiedliche Antidepressiva einschließlich deren Metaboliten messbar sind. Dazu wurde eine flüssigchromatographische Methode (HPLC) mit Ultraviolettspektroskopie (UV) entwickelt, mit der die quantitative Analyse von zehn antidepressiven und zusätzlich zwei antipsychotischen Substanzen innerhalb von 25 Minuten mit ausreichender Präzision und Richtigkeit (beide über 85%) und Sensitivität erlaubte. Durch Säulenschaltung war eine automatisierte Analyse von Blutplasma oder –serum möglich. Störende Matrixbestandteile konnten auf einer Vorsäule ohne vorherige Probenaufbereitung abgetrennt werden. Das kosten- und zeiteffektive Verfahren war eine deutliche Verbesserung für die Bewältigung von Proben im Laboralltag und damit für das TDM von Antidepressiva. Durch Analyse des klinischen Einsatzes von TDM wurden eine Reihe von Anwendungsfehlern identifiziert. Es wurde deshalb versucht, die klinische Anwendung des TDM von Antidepressiva durch die Umstellung von einer weitgehend händischen Dokumentation auf eine elektronische Bearbeitungsweise zu verbessern. Im Rahmen der Arbeit wurde untersucht, welchen Effekt man mit dieser Intervention erzielen konnte. Dazu wurde eine Labor-EDV eingeführt, mit der der Prozess vom Probeneingang bis zur Mitteilung der Messergebnisse auf die Stationen elektronisch erfolgte und die Anwendung von TDM vor und nach der Umstellung untersucht. Die Umstellung fand bei den behandelnden Ärzten gute Akzeptanz. Die Labor-EDV erlaubte eine kumulative Befundabfrage und eine Darstellung des Behandlungsverlaufs jedes einzelnen Patienten inklusive vorhergehender Klinikaufenthalte. Auf die Qualität der Anwendung von TDM hatte die Implementierung des Systems jedoch nur einen geringen Einfluss. Viele Anforderungen waren vor und nach der Einführung der EDV unverändert fehlerhaft, z.B. wurden häufig Messungen vor Erreichen des Steady State angefordert. Die Geschwindigkeit der Bearbeitung der Proben war im Vergleich zur vorher händischen Ausführung unverändert, ebenso die Qualität der Analysen bezüglich Richtigkeit und Präzision. Ausgesprochene Empfehlungen hinsichtlich der Dosierungsstrategie der angeforderten Substanzen wurden häufig nicht beachtet. Verkürzt wurde allerdings die mittlere Latenz, mit der eine Dosisanpassung nach Mitteilung des Laborbefundes erfolgte. Insgesamt ist es mit dieser Arbeit gelungen, einen Beitrag zur Verbesserung des Therapeutischen Drug Monitoring von Antidepressiva zu liefern. In der klinischen Anwendung sind allerdings Interventionen notwendig, um Anwendungsfehler beim TDM von Antidepressiva zu minimieren.

SMO inhibitor specifically targets the Hedgehog Pathway and reverts the drug-resistance of Leukemic Stem Cells

Abnormal Hedgehog signaling is associated with human malignancies. Smo, a key player of that signaling, is the most suitable target to inhibit this pathway. To this aim several molecules, antagonists of Smo, have been synthesized, and some of them have started the phase I in clinical trials. Our hospital participated to one of these studies which investigated the oral administration of a new selective inhibitor of Smo (SMOi). To evaluate ex vivo SMOi efficacy and to identify new potential clinical biomarkers of responsiveness, we separated bone marrow CD34+ cells from 5 acute myeloid leukemia (AML), 1 myelofibrosis (MF), 2 blastic phases chronic myeloid leukemia (CML) patients treated with SMOi by immunomagnetic separation, and we analysed their gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. This analysis, showed differential expression after 28 days start of therapy (p-value ≤ 0.05) of 1,197 genes in CML patients and 589 genes in AML patients. This differential expression is related to Hedgehog pathway with a p-value = 0.003 in CML patients and with a p-value = 0.0002 in AML patients, suggesting that SMOi targets specifically this pathway. Among the genes differentially expressed we observed strong up-regulation of Gas1 and Kif27 genes, which may work as biomarkers of responsiveness of SMOi treatment in CML CD34+ cells whereas Hedgehog target genes (such as Smo, Gli1, Gli2, Gli3), Bcl2 and Abca2 were down-regulated, in both AML and CML CD34+ cells. It has been reported that Bcl-2 expression could be correlated with cancer therapy resistance and that Hedgehog signaling modulate ATP-binding (ABC) cassette transporters, whose expression has been correlated with chemoresistance. Moreover we confirmed that in vitro SMOi treatment targets Hedgehog pathway, down-regulate ABC transporters, Abcg2 and Abcb1 genes, and in combination with tyrosine kinase inhibitors (TKIs) could revert the chemoresistance mechanism in K562 TKIs-resistant cell line.