Anticancer Effects of Synthetic Phosphoethanolamine on Ehrlich Ascites Tumor: An Experimental Study

Background: Antineoplastic phospholipids (ALPs) represent a promising class of drugs with a novel mode of action undergoes rapid turnover in the cell membrane of tumors, interfering with lipid signal transduction, inducing cell death. The aim of this study was to investigate the synthetic phosphoethanolamine (Pho-s) as a new anticancer agent. Materials and Methods: Cell viability and morphology were assessed by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst and rhodamine staining. Apoptosis was assessed by Annexin V and propidium iodide (PI) staining, caspase-3 activity, mitochondrial membrane potential (Delta m psi) and cell cycle analysis, combined with evaluation of tumor growth in Ehrlich Ascites Tumor (EAT) bearing mice. Results: We found that Pho-s 2.30 mg/ml induced cytotoxicity in all tumor cell lines studied without affecting normal cells. In vitro studies with EAT cells indicated that Pho-s induced apoptosis, demonstrated by an increase in Annexin-V positive cells, loss of mitochondrial potential (Delta m psi) and increased caspase-3 activity. It was also shown to increase the sub-G(1) apoptotic fraction and inhibit progression to the S phase of the cell cycle. Additionally, antitumor effects on the EAT-bearing mice showed that Pho-s, at a concentration of 35 and 70 mg/kg, inhibited tumor growth and increased the lifespan of animals without causing liver toxicity. Conclusion: These findings suggest that Pho-s is a potential anticancer candidate drug.

In vitro cytotoxicity of Selol-loaded magnetic nanocapsules against neoplastic cell lines under AC magnetic field activation

The goals of this study are to evaluate in vitro compatibility of magnetic nanomaterials and their therapeutic potential against cancer cells. Highly stable ionic magnetic fluid sample (maghemite, gamma-Fe2O3) and Selol were incorporated into polymeric nanocapsules by nanoprecipitation method. The cytotoxic effect of Selol-loaded magnetic nanocapsules was assessed on murine melanoma (B16-F10) and oral squamous cell carcinoma (OSCC) cell lines following AC magnetic field application. The influence of different nanocapsules on cell viability was investigated by colorimetric MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. In the absence of AC magnetic field Selol-loaded magnetic nanocapsules, containing 100 mu g/mL Selol plus 5 x 10(12) particle/mL, showed antitumoral activity of about 50% on B16-F10 melanoma cells while OSCC carcinoma cells demonstrated drug resistance at all concentrations of Selol and magnetic fluid (range of 100-500 mu g/mL Selol and 5 x 10(12) -2.5 x 10(13) particle/mL). On the other hand, under AC applied fields (1 MHz and 40 Oe amplitude) B16-F10 cell viability was reduced down to 40.5% (+/- 3.33) at the highest concentration of nanoencapsulated Selol. The major effect, however, was observed on OSCC cells since the cell viability drops down to about 33.3% (+/- 0.38) under application of AC magnetic field. These findings clearly indicate that the Selol-loaded magnetic nanocapsules present different toxic effects on neoplastic cell lines. Further, the cytotoxic effect was maximized under AC magnetic field application on OSCC, which emphasizes the effectiveness of the magnetohyperthermia approach. (C) 2012 American Institute of Physics. [doi: 10.1063/1.3680541]

In vitro efficacy of Coriandrum sativum, Lippia sidoides and Copaifera reticulata against Leishmania chagasi

The increased incidence of visceral leishmaniasis (VL) in Brazil is due to a lack of effective disease control measures. In addition to that, no effective treatment exists for canine VL in response to synthetic drugs. Thus, the objective of this study was to evaluate the effect of the essential oils of Coriandrum sativum and Lippia sidoides, and oleoresin from Copaifera reticulata, on Leishmania chagasi promastigotes and amastigotes. We also examined the toxicity of these treatments on the murine monocyte cell line RAW 264.7. To determine the IC50 a MTT test (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed on promastigotes, and an in situ ELISA assay was conducted on amastigotes. Here, we demonstrate that oleoresin from C. reticulata was effective against both promastigotes (IC50 of 7.88 µg.mL-1) and amastigotes (IC50 of 0.52 µg.mL-1), and neither of the two treatments differed significantly (p > 0.05) from pentamidine (IC50 of 2.149 µg.mL-1) and amphotericin B (IC50 of 9.754 µg.mL-1). Of the three plant oils tested, only oleoresin showed no toxicity toward monocyte, with 78.45% viability after treatment. Inhibition of promastigote and amastigote growth and the lack of cytotoxicity by C. reticulata demonstrate that oleoresin may be a viable option for analyzing the in vivo therapeutic effects of leishmanicidal plants

Herstellung, Struktur und Polymerisation neuer Cyclodextrin-komplexierter (Meth)acrylate und radikalische Polymerisation neuer Monomere zur Steuerung rheologischer Eigenschaften

Zusammenfassung In der vorliegenden Arbeit wurden Polymerisationseigenschaften hydrophober Monomere untersucht, die mittels methyliertem b-Cyclodextrin (me-b-CD) als Wirt/Gast-Komplexe homogen in die wäßrige Phase überführt wurden. Mit diesem Verfahren steht eine neue Variante der Polymerisation hydrophober Monomere in Wasser zur Verfügung.Die Charakterisierung der Monomer/CD-Komplexe erfolgte mittels Röntgenstrukturanalyse und 1H-NMR-Spektroskopie: steigende Größe der Gast-Komponente erhöht die Wechselwirkungen zum CD. Zur Untersuchung der Polymerisationsreaktion wurden Copolymerisationsparameter von Komplexen und Übertragungskonstanten von Mercaptoverbindungen bei Polymerisationen von Komplexen ermittelt. In Abhängigkeit der Größe der Gastkomponente und ihrer Wasserlöslichkeit resultieren unterschiedliche Reaktivitäten relativ zu Polymerisationen unkomplexierter Reaktanden in organisch-wäßriger Lösung. Außerdem wurden Copolymerisationen zwischen hydrophoben, me-b-CD-komplexierten Monomeren und wasserlöslichen Monomeren untersucht wie z.B. N-Isopropylacrylamid oder Natrium-4-(acrylamido)phenyldiazosulfonat, dessen Copolymerisation mit Styrol bislang nicht möglich war. Eine weitere Aufgabe war die Herstellung wasserstoffbrückenbindender Polymere. Hierzu wurden assoziationsfähige Monomere hergestellt und mit Methylmethacrylat copolymerisiert. Lösungen der Copolymere wurden rheologisch untersucht. Die Lösungen besitzen hohe Nullscherviskositäten bei 20°C und sind strukturviskos. Die Untersuchung der Temperaturabhängigkeit der Nullscherviskositäten ergab insbesondere bei der Lösung von Poly(N-(methacryl-2-ethyl)-N'-(3-amino-(1,2,4-triazol-2-yl))harnstoff-co-methylmethacrylat) bei niedrigen Temperaturen eine hohe Fließaktivierungsenergie, die zu höheren Temperaturen sank. Die komplexe Viskosität dieser Lösung fiel mit zunehmender Temperatur zunächst ab, stieg dann wieder an und sank erneut. Mittels DSC-Messungen konnten Phasenübergänge für dieses Fließverhalten verantwortlich gemacht werden. Außerdem kann Poly(N-(methacryl-2-ethyl)-N'-(3-amino-(1,2,4-triazol-2-yl))harnstoff-co-methylmethacrylat) thermisch vernetzen.

Kontrollierte radikalische Polymerisation von polaren Monomeren

In Rahmen dieser Arbeit wurden neben dem 1,3,5,5-Tetraphenyl-D3-1,2,4-triazolin-2-yl (1) zwei neue Triazolinyl-Derivate synthetisiert: 3-(4-Biphenylyl)-1,5,5-triphenyl-D3-1,2,4-triazolin-2-yl (2) und 3-(4-Cyanophenyl)-1,5,5-triphenyl-D3-1,2,4-triazolin-2-yl (3). Diese sind bei Raumtemperatur stabil und können das Gleichgewicht der radikalischen Polymerisation steuern, ohne sich vollständig zu zersetzen. Sie neigen jedoch bei erhöhter Temperatur zur langsamen Zersetzung und Freisetzung eines Phenylradikals.Die Triazolinyle 1-3 wurden in der radikalischen Polymerisation sowohl mit polaren Monomeren wie 2-Vinylpyridin (2VP), Methylmethacrylat (MMA), Ethylmethacrylat (EMA) und N,N-Dimethylaminoethylmethacrylat (DMAEMA) als auch mit dem unpolaren Monomer Styrol (St) umgesetzt. Triazolinyl 1 wurde in der Polymerisation von Vinylacetat und Acrylnitril eingesetzt. Die Polymerisation von Natrium-Styrolsulfonat in Wasser wurde in Gegenwart von Cyano-Triazolinyl 3 in Form eines Komplexes mit Cyclodextrin durchgeführt.Der Einsatz der Triazolinyle 1-3 in der Polymerisation von Styrol, 2-Vinylpyridin, Methylmethacrylat, Ethylmethacrylat, N,N-dimethylaminoethylmethacrylat führte zu einem kontrollierten Prozess. Bei der Polymerisation von Vinylacetat und Acrylnitril konnte man durch den Einsatz von Triazolinyl 1 zwar den Monomerverbrauch und das Molekulargewicht kontrollieren, es wurden aber breite Molekulargewichtsverteilungen erhalten, die aus den irreversiblen Abbruchreaktionen resultierten. Im Gegensatz dazu konnten in der Polymerisation von Natrium-Styrolsulfonat weder die Umsätze noch die Molekulargewichte gesteuert werden. Das sterisch anspruchvolle Additiv ermöglichte keine Einstellung des kontrollierenden Gleichgewichts.Die Triazolinyl-endfunktionalisierten Polymere wurden als Makroinitiator in Gegenwart anderer Monomere eingesetzt. Dabei wurden Blockcopolymere erhalten. So gelang die Synthese von verschiedenen Blockstrukturen, die sonst nur unter anionischer oder Gruppentransfer-Polymerisation zugänglich sind.

Conjugated semiconducting organic materials for electronic applications

Since conjugated polymers, i.e. polymers with spatially extended pi-bonding system have offered unique physical properties, unobtainable for conventional polymers, significant research efforts directed to better understanding of their chemistry, physics and engineering have been undertaken in the past two and half decades. In this thesis we discuss the synthesis, characterisation and investigation of conjugated semiconducting organic materials for electronic applications. Owing to the versatile properties of metal-organic hybrid materials, there is significant promise that these materials can find use in optical or electronic devices in the future. In addressing this issue, the synthesis of bisthiazol-2-yl-amine (BTA) based polymers is attempted and their metallation is investigated. The focus of this work has been to examine whether the introduction of coordinating metal ions onto the polymer backbone can enhance the conductivity of the material. These studies can provide a basis for understanding the photophysical properties of metal-organic polymers based on BTA. In their neutral (undoped) form conjugated polymers are semiconductors and can be used as active components of plastics electronics such as polymer light-emitting diodes, polymer lasers, photovoltaic cells, field-effect transistors, etc. Toward this goal, it is an objective of the study to synthesize and characterize new classes of luminescent polymeric materials based on anthracene and phenanthrene moieties. A series of materials based on polyphenylenes and poly(phenyleneethynylene)s with 9,10-anthrylene subunits are not only presented but the synthesis and characterization of step-ladder and ladder poly(p-phenylene-alt-anthrylene)s containing 9,10-anthrylene building groups within the main chain are also explored. In a separate work, a series of soluble poly-2,7- and 3,6-phenanthrylenes are synthesized. This can enable us to do a systematic investigation into the optical and electronic properties of PPP-like versus PPV-like. Besides, the self-organization of 3,6-linked macrocyclic triphenanthrylene has been investigated by 2D wide-angle X-ray scattering experiments performed on extruded filaments in solution and in the bulk. Additionally, from the concept that donor-acceptor materials can induce efficient electron transfer, the covalent incorporation of perylene tetracarboxydiimide (PDI) into one block of a poly(2,7-carbazole) (PCz)-based diblock copolymer and 2,5-pyrrole based on push-pull type material are achieved respectively.

Die Vielseitigkeit von Carbazol in pi-konjugierten Materialien: Makrozyklen, Doppelstrang-Strukturen und Donor-Akzeptor-Copolymere

In dieser Arbeit wurden die vielfältigen Anwendungsmöglichkeiten von Carbazol-reichen Verbindungen in unterschiedlichen Architekturen von pi-konjugierten Materialien untersucht. Die Darstellung der Makrozyklen in Kapitel 1 erfolgte über eine templatunterstützte Synthese, da die ausschließlich über ihre para-Position verknüpften Einheiten einen Zugang über ihre Geometrie nicht erlaubten. Cyclododeca-2,7-fluoren wurde als zyklische Modellverbindung für die zahlreichen bekannten linearen Oligo- und Polyfluorene dargestellt, um den Einfluss der Ringstruktur auf die optischen Eigenschaften dieser blau emittierenden Verbindungen zu untersuchen. Motiviert von den geringen Ausbeuten bei der Zyklisierung des Rings unter Yamamoto-Bedingungen wurde nach einer alternativen Kupplungsreaktion gesucht, die den Zugang zu größeren Substanzmengen erlaubt. Die Einführung von terminalen Ethinylgruppen an die zu verknüpfenden Bausteine erlaubte über die Glaser-Eglinton-Reaktion nicht nur eine Verbesserung der Reaktionsausbeute um den Faktor 10, sondern gleichzeitig auch die Erweiterung des Durchmessers auf rund 5 nm. Demonstriert wurde dies anhand der Darstellung von Cyclo[tetrakis(2,7-diethinyltriscarbazol)] und Cyclo[tetrakis(2,7-diethinyltrisfluoren)].rnDer zweite Abschnitt der Dissertation befasste sich mit ausgedehnten zweidimensionalen Strukturen, zu denen auch mehrsträngige Polymere zählen. Für die Darstellung eines Doppelstrang-Polymers mit hoher Periodizität in den beiden in regelmäßigen Abständen verknüpften Strängen wurde ein neues Konzept entwickelt, dass den Zugang zu solchen Verbindungen erlauben sollte. Dieses basierte auf einem vierfach funktionalisierten Carbazol-Makromonomer. Die Polymerisationen wurden in zwei Schritten über Glaser-Eglinton-Reaktionen durchgeführt. Um unkontrollierte Kupplungen zu vermeiden, wurden zwei der vier reaktiven Positionen am Monomer zunächst über Schutzgruppen maskiert und nach der ersten Polymerisation wieder aktiviert. Neben diesem Ansatz über oxidative Ethinylkupplungen wurde auch eine Organometall-Variante mit einem Platin-Komplex im Polymerrückgrat vorgestellt. rnIm dritten Kapitel wurde die Darstellung von Donor-Akzeptor-Polymeren mit niedriger Bandlücke für den Einsatz in organischen Solarzellen besprochen. Im Gegensatz zu den bisherigen Ansätzen in der Literatur wurden hier ganz gezielt Donor- und Akzeptor-Einheiten mit einer Länge von mindestens 1,5 nm synthetisiert und kombiniert, um zu untersuchen, welche Auswirkungen der Einsatz solcher Makromonomere auf die Eigenschaften der Materialien hat. Dabei bestand der Akzeptor-Baustein in drei Polymeren aus Bis[2,7-di-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9,18,18-tetraoctylindeno[1,2-b]-yl]-indeno[1,2-b]fluoren-12,15-dion, während die Donor-Stärke durch den Übergang von einem reinen leiterverbrückten Carbazol-Monomer über eine gemischte Carbazol-Thiophen-Einheit hin zu einem reinen Thiophen-Precursor variiert wurde. Dabei zeigte das Polymer mit Thiophen als Donor den höchsten Zellwirkungsgrad von 0,41 %.rn

Photo-induced reversible assembly of colloidal polymer nanoparticles

Polymer nanoparticles functionalized on the surface with photo-responsive labels were synthesized. In a first synthetic step, polystyrene was copolymerized with the cross-linker divinylbenzene and poly(ethylene glycol) acrylate in a miniemulsion, to produce nano-sized spheres (~ 60 nm radius) with terminal hydroxyl groups, which were functionalized in a subsequent synthetic step with photo-responsive labels. For this purpose, two photo-active molecular structures were separately used: anthracene, which is well known to form covalently bonded dimers upon photo-excitation; and pyrene, which only forms short lived excited state dimers (excimers). Acid derivatives of these labels (9-anthracene carboxylic acid and 1-pyrene butyric acid) were bonded to the hydroxyl terminal groups of the nanoparticles through an esterification reaction, via the intermediate formation of the corresponding acid chloride.rnThe obtained labeled nanoparticles appeared to be highly hydrophobic structures. They formed lyophobic suspensions in water, which after analysis by dynamic light scattering (DLS) and ultramicroscopic particle tracking, appeared to equilibrate as a collection of singly dispersed nanoparticles, together with a few nanoparticle aggregates. The relative amount of aggregates decreased with increasing amounts of the surfactant sodium dodecyl sulfate (SDS), thus confirming that aggregation is an equilibrated state resulting from lyophobicity. The formation of such aggregates was corroborated using scanning electron microscopy (SEM). The photo-irradiation of the lyophobic aqueous suspensions of anthracene labeled nanoparticles (An-NP) resulted in the formation of higher aggregates, as evidenced by DLS and ultramicroscopy. The obtained state of aggregation could be reverted by sonication. The possibility to re-aggregate the system in subsequent photo-excitation and sonication cycles was established. Likewise, the photo-irradiation of lyophobic aqueous suspensions of pyrene-labeled nanoparticles (Py-NP) resulted in the formation of higher aggregates, as evidenced by DLS and ultramicroscopy. These appeared to remain aggregated due to hydrophobic interactions. This system could also be re-dispersed by sonication and re-aggregated in subsequent cycles of photo-excitation and sonication.

Control over microstructure evolution and device performance in solution processable organic field-effect transistors

Die Arbeit beschäftigt sich mit der Kontrolle von Selbstorganisation und Mikrostruktur von organischen Halbleitern und deren Einsatz in OFETs. In Kapiteln 3, 4 und 5 eine neue Lösungsmittel-basierte Verabeitungsmethode, genannt als Lösungsmitteldampfdiffusion, ist konzipiert, um die Selbstorganisation von Halbleitermolekülen auf der Oberfläche zu steuern. Diese Methode als wirkungsvolles Werkzeug erlaubt eine genaue Kontrolle über die Mikrostruktur, wie in Kapitel 3 am Beispiel einer D-A Dyad bestehend aus Hexa-peri-hexabenzocoronene (HBC) als Donor und Perylene Diimide (PDI) als Akzeptor beweisen. Die Kombination aus Oberflächenmodifikation und Lösungsmitteldampf kann die Entnetzungseffekte ausgleichen, so dass die gewüschte Mikrostruktur und molekulare Organisation auf der Oberfläche erreicht werden kann. In Kapiteln 4 und 5 wurde diese Methode eingesetzt, um die Selbstorganisation von Dithieno[2, 3-d;2’, 3’-d’] benzo[1,2-b;4,5-b’]dithiophene (DTBDT) und Cyclopentadithiophene -benzothiadiazole copolymer (CDT-BTZ) Copolymer zu steuern. Die Ergebnisse könnten weitere Studien stimulieren und werfen Licht aus andere leistungsfaähige konjugierte Polymere. rnIn Kapiteln 6 und 7 Monolagen und deren anschlieβende Mikrostruktur von zwei konjugierten Polymeren, Poly (2,5-bis(3-alkylthiophen-2-yl)thieno[3,2-b]thiophene) PBTTT und Poly{[N,N ′-bis(2-octyldodecyl)-naphthalene-1,4,5,8-bis (dicarboximide)-2,6-diyl]-alt-5,5′- (2,2′-bithiophene)}, P(NDI2OD-T2)) wurden auf steife Oberflächen mittels Tauchbeschichtung aufgebracht. Da sist das erste Mal, dass es gelungen ist, Polymer Monolagen aus der Lösung aufzubringen. Dieser Ansatz kann weiter auf eine breite Reihe von anderen konjugierten Polymeren ausgeweitet werden.rnIn Kapitel 8 wurden PDI-CN2 Filme erfolgreich von Monolagen zu Bi- und Tri-Schichten auf Oberflächen aufgebracht, die unterschiedliche Rauigkeiten besitzen. Für das erste Mal, wurde der Einfluss der Rauigkeit auf Lösungsmittel-verarbeitete dünne Schichten klar beschrieben.rn

Characterization of HgCl2 Tridentate Amine Complexes by X-ray Crystallography, NMR and ESI-MS

Two new HgCl2 complexes of tridentate nitrogen ligands were characterized by X-ray crystallography, proton NMR spectroscopy and ESI-MS. The five-coordinate complex [Hg(BMPA)Cl-2] (1) (BMPA = bis(2-pyridylmethyl)amine) crystallized from acetonitrile/m-xylene by slow evaporation in the monoclinic space group P2(1)/n with a = 8.3896(8) , b = 12.8020(13) , c = 13.3526(13) , alpha = 90A degrees, beta A = 90.480(2)A degrees, gamma A = 90A degrees and z = 4. The square pyramidal structure (tau = 0.009) has approximate C (s) symmetry. Despite comparable Hg-N bond lengths in 1, inversion of the central nitrogen was rapid on the chemical shift time scale in dilute solution except at very low temperatures. The related complex [Hg(BEPA)Cl-2] (2) (BEPA = bis(2-{pyrid-2-yl}ethyl)amine) crystallized from acetonitrile/ethyl acetate/hexanes by slow diffusion in the orthorhombic space group Pnma with a = 13.424(3) , b = 14.854(3) , c = 8.118(2) , alpha = 90A degrees, beta A = 90A degrees, gamma A = 90A degrees and z = 4. The mixed geometry structure (tau = 0.56) also has crystallographic mirror symmetry as well as C (s) point group symmetry. In dilute acetonitrile solution, 1 was stable while 2 slowly converted to a more thermodynamically stable complex.

The Use Of Amido- And Imido-Functionalized Alpha Hydroxy Acids In The Creation Of Biodegradable Polyesters: An Exploration Of Va

The purpose of this thesis was to synthesize biodegradable polyesters from a wide array of functionalized ¿-hydroxy acids. The initial strategy was to use amido-functionalized ¿-hydroxy acids and 2-bromopropanoyl bromide to form amido-functionalized cyclic diesters. Then, the resulting cyclic diesters would be used in ring opening polymerization to create biodegradable polyesters. However, the spontaneous rapid degradation of the secondary amido-functionalized cyclic diester structure, as seen with 2-benzamido-hydroxyacetic acid, limited ring formation to tertiary amido-functionalized ¿-hydroxy acids. Also, the hydrophilic nature of most ¿-hydroxy acids allowed water into the crystal structure of the ¿-hydroxy acid. Then, when the ¿-hydroxy acid was used in ring forming reactions, the associated water deactivated reactive reagents and limited cyclic diester synthesis. These issues led to the synthesis of hydrophobic and tertiary amido- and imido-functionalized ¿-hydroxy acids, 2-phthalimido-2-hydroxyacetic acid and 2-(1-oxoisoindolin-2-yl) hydroxyacetic acid. The new ¿-hydroxy acids were used in two new polymerization techniques, melt polycondensation and solution polymerization, instead of ring open polymerization. Melt polycondensation and solution polymerization had shown previous success in forming oligomers of amido-functionalized ¿-hydroxy acids. Melt polycondensation was conducted by heating the monomer past its melting temperature under reduced pressure. The uncatalyzed melt polycondensation of 2-(1-oxoisoindolin-2-yl) hydroxyacetic acid created polyesters (¿ 960 g/mol). The scandium(III) trifluoromethanesulfonate enhanced melt polycondensation polymerization created slightly larger oligomers (¿ 1340 g/mol). However, 2-phthalimido-2-hydroxyacetic acid was not compatible with melt polycondensation because thermal degradation occurred. Thus, solution polymerization was conducted via Steglich esterification. Only oligomeric functionalized polyesters were formed (¿ 1060 g/mol). Future work should focus on optimization of the catalyst and the reaction conditions to obtain higher molecular weight polyesters. Also, 2-(1-oxoisoindolin-2-yl) hydroxyacetic acid should be utilized in the cyclic diester synthesis technique.

Prostate specific antigen expression does not necessarily correlate with prostate cancer cell growth

PURPOSE: The antiproliferative effects of pharmacological agents used for androgen ablative therapy in prostate cancer, including goserelin, bicalutamide and cyproterone acetate (Fluka Chemie, Buchs, Switzerland), were tested in vitro. It was determined whether they affected prostate specific antigen mRNA and protein expression independent of growth inhibition. MATERIALS AND METHODS: Goserelin, bicalutamide (AstraZeneca, Zug, Switzerland) and cyproterone acetate were added to prostate specific antigen expressing, androgen dependent LNCaP and androgen independent C4-2 cell line (Urocor, Oklahoma City, Oklahoma) cultures. Proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay (Roche, Mannheim, Germany). Prostate specific antigen mRNA expression was assessed by quantitative real-time polymerase chain reaction. Secreted prostate specific antigen protein levels were quantified by microparticle enzyme-immunoassay. RESULTS: Goserelin inhibited cell growth and prostate specific antigen protein secretion in LNCaP and C4-2 cells. Prostate specific antigen mRNA expression was not decreased. Bicalutamide did not affect cell growth or prostate specific antigen mRNA expression in LNCaP or C4-2 cells, although it significantly decreased prostate specific antigen protein secretion in LNCaP and to a lesser extent in C4-2 cells. Cyproterone acetate decreased the growth of C4-2 but not of LNCaP cells. It did not affect prostate specific antigen mRNA or protein expression in either cell line. CONCLUSIONS: Prostate specific antigen expression does not necessarily correlate with cell growth. Without a substantial effect on cell growth bicalutamide lowers prostate specific antigen synthesis, whereas cyproterone acetate decreases cell growth with no effect on prostate specific antigen secretion. Prostate specific antigen expression may be influenced by growth inhibition but also by altered mRNA and protein levels depending on the agent, its concentration and the cell line evaluated. For interpreting clinical trials prostate specific antigen is not necessarily a surrogate end point marker for a treatment effect on prostate cancer cell growth.

Differential response of porcine osteoblasts and chondrocytes in cell or tissue culture after 5-aminolevulinic acid-based photodynamic therapy

OBJECTIVE: Outcome in osteochondral allografting is limited by the immunological incompatibility of the grafted tissue. Based on a resistance of chondrocytes to photodynamic therapy in cell culture it is proposed that 5-aminolevulinic acid-based photodynamic therapy (5-ALA-PDT) might be used to inactivate bone while maintaining viability of chondrocytes and thus immunomodulate bone selectively. METHODS: Chondrocytes and osteoblasts from porcine humeral heads were either isolated (cell culture) or treated in situ (tissue culture). To quantify cytotoxic effects of 5-ALA-PDT (0-20J/cm(2), 100mW/cm(2)) an (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide) (MTT)-assay was used in cell culture and in situ hybridization in tissue culture to assess metabolic active cells (functional osteoblasts: colalpha(1)(I) mRNA, functional chondrocytes: colalpha(1)(II) mRNA). RESULTS: In cell culture, survival after 5-ALA-PDT was significantly higher for chondrocytes (5J/cm(2): 87+/-12% compared to untreated cells) than for osteoblasts (5J/cm(2): 12+/-11%). In tissue culture, the percentage of functional chondrocytes in cartilage showed a decrease after 5-ALA-PDT (direct fixation: 92+/-2%, 20J/cm(2): 35+/-15%; P<0.0001). A significant decrease in the percentage of bone surfaces covered by functional osteoblasts was observed in freshly harvested (31+/-3%) compared to untreated tissues maintained in culture (11+/-4%, P<0.0001), with no further decrease after 5-ALA-PDT. CONCLUSION: Chondrocytes were more resistant to 5-ALA-PDT than osteoblasts in cell culture, while in tissue culture a loss of functional chondrocytes was observed after 5-ALA-PDT. Since osteoblasts - but not chondrocytes - were sensitive to the tissue culture conditions, devitalized bone with functional cartilage might already be achieved by applying specific tissue culture conditions even without 5-ALA-PDT.

Direct in vitro and in vivo comparison of 161Tb and 177Lu using a tumour-targeting folate conjugate

Purpose The radiolanthanide 161Tb (T 1/2 = 6.90 days, Eβ− av = 154 keV) was recently proposed as a potential alternative to 177Lu (T 1/2 = 6.71 days, Eβ− av = 134 keV) due to similar physical decay characteristics but additional conversion and Auger electrons that may enhance the therapeutic efficacy. The goal of this study was to compare 161Tb and 177Lu in vitro and in vivo using a tumour-targeted DOTA-folate conjugate (cm09). Methods 161Tb-cm09 and 177Lu-cm09 were tested in vitro on folate receptor (FR)-positive KB and IGROV-1 cancer cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. In vivo 161Tb-cm09 and 177Lu-cm09 (10 MBq, 0.5 nmol) were investigated in two different tumour mouse models with regard to the biodistribution, the possibility for single photon emission computed tomography (SPECT) imaging and the antitumour efficacy. Potentially undesired side effects were monitored over 6 months by determination of plasma parameters and examination of kidney function with quantitative SPECT using 99mTc-dimercaptosuccinic acid (DMSA). Results To obtain half-maximal inhibition of tumour cell viability a 4.5-fold (KB) and 1.7-fold (IGROV-1) lower radioactivity concentration was required for 161Tb-cm09 (IC50 ~0.014 MBq/ml and ~2.53 MBq/ml) compared to 177Lu-cm09 (IC50 ~0.063 MBq/ml and ~4.52 MBq/ml). SPECT imaging visualized tumours of mice with both radioconjugates. However, in therapy studies 161Tb-cm09 reduced tumour growth more efficiently than 177Lu-cm09. These findings were in line with the higher absorbed tumour dose for 161Tb-cm09 (3.3 Gy/MBq) compared to 177Lu-cm09 (2.4 Gy/MBq). None of the monitored parameters indicated signs of impaired kidney function over the whole time period of investigation after injection of the radiofolates. Conclusion Compared to 177Lu-cm09 we demonstrated equal imaging features for 161Tb-cm09 but an increased therapeutic efficacy for 161Tb-cm09 in both tumour cell lines in vitro and in vivo. Further preclinical studies using other tumour-targeting radioconjugates are clearly necessary to draw final conclusions about the future clinical perspectives of 161Tb.

Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: from ultrapotent to hyperpotent.

Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho=0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC50 values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.