A "Trojan horse" strategy to reverse drug-resistance in brain tumors

Los gliomas malignos representan una de las formas más agresivas de los tumores del sistema nervioso central (SNC). De acuerdo con la clasificación de los tumores cerebrales de la Organización Mundial de la Salud (OMS), los astrocitomas han sido categorizados en cuatro grados, determinados por la patología subyacente. Es así como los gliomas malignos (o de alto grado) incluyen el glioma anaplásico (grado III) así como el glioblastoma multiforme (GBM, grado IV),estos últimos los más agresivos con el peor pronóstico (1). El manejo terapéutico de los tumores del SNC se basa en la cirugía, la radioterapia y la quimioterapia, dependiendo de las características del tumor, el estadio clínico y la edad (2),(3), sin embargo ninguno de los tratamientos estándar es completamente seguro y compatible con una calidad de vida aceptable (3), (4). En general, la quimioterapia es la primera opción en los tumores diseminados, como el glioblastoma invasivo y el meduloblastoma de alto riesgo o con metástasis múltiple, pero el pronóstico en estos pacientes es muy pobre (2),(3). Solamente nuevas terapias dirigidas (2) como las terapias anti-angiogénicas (4); o terapias génicas muestran un beneficio real en grupos limitados de pacientes con defectos moleculares específicos conocidos (4). De este modo, se hace necesario el desarrollo de nuevas terapias farmacológicas para atacar los tumores cerebrales. Frente a las terapias los gliomas malignos son con frecuencia quimioresistentes, y esta resistencia parece depender de al menos dos mecanismos: en primer lugar, la pobre penetración de muchas drogas anticáncer a través de la barrera hematoencefálica (BBB: Blood Brain Barrier), la barrera del fluido sangre-cerebroespinal (BCSFB: Blood-cerebrospinal fluid barrier) y la barrera sangre-tumor (BTB: blood-tumor barrier). Dicha resistencia se debe a la interacción de la droga con varios transportadores o bombas de eflujo de droga ABC (ABC: ATP-binding cassette) que se sobre expresan en las células endoteliales o epiteliales de estas barreras. En segundo lugar, estos transportadores de eflujo de drogas ABC propios de las células tumorales confieren un fenotipo conocido como resistencia a multidrogas (MDR: multidrug resistance), el cual es característico de varios tumores sólidos. Este fenotipo también está presente en los tumores del SNC y su papel en gliomas es objeto de investigación (5). Por consiguiente el suministro de medicamentos a través de la BBB es uno de los problemas vitales en los tratamientos de terapia dirigida. Estudios recientes han demostrado que algunas moléculas pequeñas utilizadas en estas terapias son sustratos de la glicoproteína P (Pgp: P-gycoprotein), así como también de otras bombas de eflujo como las proteínas relacionadas con la resistencia a multidrogas (MRPs: multidrug resistance-related proteins (MRPs) o la proteína relacionada con cáncer de seno (BCRP: breast-cancer resistance related protein)) que no permiten que las drogas de este tipo alcancen el tumor (1). Un sustrato de Pgp y BCRP es la DOXOrubicina (DOXO), un fármaco utilizado en la terapia anti cáncer, el cual es muy eficaz para atacar las células del tumor cerebral in vitro, pero con un uso clínico limitado por la poca entrega a través de la barrera hematoencefálica (BBB) y por la resistencia propia de los tumores. Por otra parte las células de BBB y las células del tumor cerebral tienen también proteínas superficiales, como el receptor de la lipoproteína de baja densidad (LDLR), que podría utilizarse como blanco terapéutico en BBB y tumores cerebrales. Es asi como la importancia de este estudio se basa en la generación de estrategias terapéuticas que promuevan el paso de las drogas a través de la barrera hematoencefalica y tumoral, y a su vez, se reconozcan mecanismos celulares que induzcan el incremento en la expresión de los transportadores ABC, de manera que puedan ser utilizados como blancos terapéuticos.Este estudio demostró que el uso de una nueva estrategia basada en el “Caballo de Troya”, donde se combina la droga DOXOrubicina, la cual es introducida dentro de un liposoma, salvaguarda la droga de manera que se evita su reconocimiento por parte de los transportadores ABC tanto de la BBB como de las células del tumor. La construcción del liposoma permitió utilizar el receptor LDLR de las células asegurando la entrada a través de la BBB y hacia las células tumorales a través de un proceso de endocitosis. Este mecanismo fue asociado al uso de estatinas o drogas anticolesterol las cuales favorecieron la expresión de LDLR y disminuyeron la actividad de los transportadores ABC por nitración de los mismos, incrementando la eficiencia de nuestro Caballo de Troya. Por consiguiente demostramos que el uso de una nueva estrategia o formulación denominada ApolipoDOXO más el uso de estatinas favorece la administración de fármacos a través de la BBB, venciendo la resistencia del tumor y reduciendo los efectos colaterales dosis dependiente de la DOXOrubicina. Además esta estrategia del "Caballo de Troya", es un nuevo enfoque terapéutico que puede ser considerado como una nueva estrategia para aumentar la eficacia de diferentes fármacos en varios tumores cerebrales y garantiza una alta eficiencia incluso en un medio hipóxico,característico de las células cancerosas, donde la expresión del transportador Pgp se vió aumentada. Teniendo en cuenta la relación entre algunas vías de señalización reconocidas como moduladores de la actividad de Pgp, este estudio presenta no solo la estrategia del Caballo de Troya, sino también otra propuesta terapéutica relacionada con el uso de Temozolomide más DOXOrubicina. Esta estrategia demostró que el temozolomide logra penetrar la BBB por que interviene en la via de señalización de la Wnt/GSK3/β-catenina, la cual modula la expresión del transportador Pgp. Se demostró que el TMZ disminuye la proteína y el mRNA de Wnt3 permitiendo plantear la hipótesis de que la droga al disminuir la transcripción del gen Wnt3 en células de BBB, incrementa la activación de la vía fosforilando la β-catenina y conduciendo a disminuir la β-catenina nuclear y por tanto su unión al promotor del gen mdr1. Con base en los resultados este estudio permitió el reconocimiento de tres mecanismos básicos relacionados con la expresión de los transportadores ABC y asociados a las estrategias empleadas: el primero fue el uso de las estatinas, el cual condujo a la nitración de los transportadores disminuyendo su actividad por la via del factor de transcripción NFκB; el segundo a partir del uso del temozolomide, el cual metila el gen de Wnt3 reduciendo la actividad de la via de señalización de la la β-catenina, disminuyendo la expresión del transportador Pgp. El tercero consistió en la determinación de la relación entre el eje RhoA/RhoA quinasa como un modulador de la via (no canónica) GSK3/β-catenina. Se demostró que la proteína quinasa RhoA promovió la activación de la proteína PTB1, la cual al fosforilar a GSK3 indujo la fosforilación de la β-catenina, lo cual dio lugar a su destrucción por el proteosoma, evitando su unión al promotor del gen mdr1 y por tanto reduciendo su expresión. En conclusión las estrategias propuestas en este trabajo incrementaron la citotoxicidad de las células tumorales al aumentar la permeabilidad no solo de la barrera hematoencefálica, sino también de la propia barrera tumoral. Igualmente, la estrategia del “Caballo de Troya” podría ser útil para la terapia de otras enfermedades asociadas al sistema nervioso central. Por otra parte estos estudios indican que el reconocimiento de mecanismos asociados a la expresión de los transportadores ABC podría constituir una herramienta clave en el desarrollo de nuevas terapias anticáncer.

Polymer-mediated disruption of drug crystallinity

Ibuprofen (IB), a BCS Class II compound, is a highly crystalline substance with poor solubility properties. Here we report on the disruption of this crystalline structure upon intimate contact with the polymeric carrier cross-linked polyvinylpyrrolidone (PVP-CL) facilitated by low energy simple mixing. Whilst strong molecular interactions between APIs and carriers within delivery systems would be expected on melting or through solvent depositions, this is not the case with less energetic mixing. Simple mixing of the two compounds resulted in a significant decrease in the differential scanning calorimetry (DSC) melting enthalpy for IB, indicating that approximately 30% of the crystalline content was disordered. This structural change was confirmed by broadening and intensity diminution of characteristic IB X-ray powder diffractometry (PXRD) peaks. Unexpectedly, the crystalline content of the drug continued to decrease upon storage under ambient conditions. The molecular environment of the mixture was further investigated using Fourier transform infrared (FT-IR) and Fourier transform Raman (FT-Raman) spectroscopy. These data suggest that the primary interaction between these components of the physical mix is hydrogen bonding, with a secondary mechanism involving electrostatic/hydrophobic interactions through the IB benzene ring. Such interactions and subsequent loss of crystallinity could confer a dissolution rate advantage for IB. (C) 2006 Elsevier B.V. All rights reserved.

Synthesis and biological in vitro evaluation of novel PEG-psoralen conjugates

Psoralens are well-known photosensitizers, and 8- methoxypsoralen and 4,5',8-trimethylpsoralen are widely used in photomedicine as "psoralens plus UVA therapy" (PUVA), in photopheresis, and in sterilization of blood preparations. In an attempt to improve the therapeutic efficiency of PUVA therapy and photopheresis, four poly(ethylene glycol) (PEG)-psoralen conjugates were synthesized to promote tumor targeting by the enhanced permeability and retention (EPR) effect. Peptide linkers were used to exploit specific enzymatic cleavage by lysosomal proteases. A new psoralen, 4-hydroxymethyl-4', 8-dimethylpsoralen (6), suitable for polymer conjugation was synthesized. The hydroxy group allowed exploring different strategies for PEG conjugation, and linkages with different stability such ester or urethanes were obtained. PEG (5 kDa) was covalently conjugated to the new psoralen derivative using four different linkages, namely, (i) direct ester bond (7), (ii) ester linkage with a peptide spacer (8), (iii) a carbamic linker (9), and (iv) a carbamic linker with a peptide spacer (12). The stability of these new conjugates was assessed at different pHs, in plasma and following incubation with cathepsin B. Conjugates 7 and 8 were rapidly hydrolyzed in plasma, while 9 was stable in buffer and in the presence of cathepsin B. As expected, only the conjugates containing the peptide linker released the drug in presence of cathepsin B. In vitro evaluation of the cytotoxic activity in the presence and absence of light was carried out in two cell lines (MCF-7 and A375 cells). Conjugates 7 and 8 displayed a similar activity to the free drug (probably due to the low stability of the ester linkage). Interestingly, the conjugates containing the carbamate linkage (9 and 12) were completely inactive in the dark (IC50 > 100 mu M in both cell lines). However, antiproliferative activity become apparent after UV irradiation. Conjugate 12 appears to be the most promising for future in vivo evaluation, since it was relatively stable in plasma, which should allow tumor targeting and drug release to occur by cathepsin B-mediated hydrolysis.

Polysialic acid as a drug carrier: evaluation of a new polysialic acid–epirubicin conjugate and its comparison against established drug carriers

This paper explores the potential of polysialic acid (PSA) as a carrier for low molecular weight anticancer drugs. A PSA–epirubicin (Epi) conjugate was synthesized and compared against Epi conjugates containing established carriers, namely: N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, poly(ethylene glycol) (PEG) and polyglutamic acid (PGA). Biological assessments in the breast cancer cell line MCF-7 and in the anthracycline resistant MCF-7/DX showed that the PSA–Epi conjugate had the highest activity (40% and 30% cell death in the two cell lines at 1 mM Epi equiv., respectively). FACS studies confirmed internalization of all conjugates by cholesterol-dependent endocytosis. PSA–Epi showed release of Epi (40% at 5 h) when incubated with lysosome extracts. In vivo evaluation showed that all conjugates had a significantly longer half-life compared to free Epi. This study also allowed an investigation on the effect of the polymeric carrier on the biological activity of a conjugate, with the biodegradability of the carrier emerging as an important feature.

HPMA copolymer-aminoglutethimide conjugates inhibit aromatase in MCF-7 cell lines

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer–doxorubicin (Dox) has already shown clinical activity in breast cancer patients. Moreover, we have recently found that an HPMA conjugate containing a combination of both Dox and the aromatase inhibitor aminoglutethimide (AGM) shows significantly increased anti-tumour activity in vitro. To better understand the mechanism of action of HPMA copolymer–AGM conjugates several models were used here to investigate their effect on cell growth and aromatase inhibition. Cytotoxicity of HPMA copolymer conjugates containing AGM, Dox and also the combination AGM–Dox was determined by MTT assay in MCF-7 and MCF-7ca cells. Androstenedione (5 × 10− 8 M) stimulates the growth of MCF-7ca cells. Both free AGM and polymer-bound AGM (0.2–0.4 mg/ml) were shown to block this mitogenic activity. When MCF-7ca cells were incubated [3H]androstenedione both AGM and HPMA copolymer–GFLG–AGM (0.2 mg/ml AGM-equiv.) showed the ability to inhibit aromatase. Although, free AGM was able to inhibit isolated human placental microsomal aromatase in a concentration dependent manner, polymer-bound AGM was not, suggesting that drug release is essential for activity of the conjugate. HPMA copolymer conjugates containing aromatase inhibitors have potential for the treatment of hormone-dependant cancers, and it would be particularly interesting to explore further as potential therapies in post-menopausal women as components of combination therapy.

Xylan from corn cobs, a promising polymer for drug delivery: Production and characterization

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Modeling a system of phosphated cross-linked high amylose for controlled drug release. Part 1: Synthesis and polymer characterization

High amylose was cross-linked with sodium trimetaphosphate (STMP) using 2% and 4% solutions of NaOH at room temperature with reaction contact times of 0.5, 1, 2 and 4 h. The different polymers obtained were analyzed by FT IR, C-13 and P-31 solid state NMR, SEM and C, H and P elemental analysis. The results were used to propose a two-stage mechanism for phosphate incorporation, the first being kinetically controlled. (C) 2008 Elsevier Ltd. All rights reserved.

Biodegradable polymer drug-eluting stents reduce the risk of stent thrombosis at 4 years in patients undergoing percutaneous coronary intervention: a pooled analysis of individual patient data from the ISAR-TEST 3, ISAR-TEST 4, and LEADERS randomized trials

The efficacy of durable polymer drug-eluting stents (DES) is delivered at the expense of delayed healing of the stented vessel. Biodegradable polymer DES aim to avoid this shortcoming and may potentially improve long-term clinical outcomes, with benefit expected to accrue over time. We sought to compare long-term outcomes in patients treated with biodegradable polymer DES vs. durable polymer sirolimus-eluting stents (SES).

Long-term outcomes of biodegradable polymer versus durable polymer drug-eluting stents in patients with diabetes a pooled analysis of individual patient data from 3 randomized trials

BACKGROUND There is ongoing debate on the optimal drug-eluting stent (DES) in diabetic patients with coronary artery disease. Biodegradable polymer drug-eluting stents (BP-DES) may potentially improve clinical outcomes in these high-risk patients. We sought to compare long-term outcomes in patients with diabetes treated with biodegradable polymer DES vs. durable polymer sirolimus-eluting stents (SES). METHODS We pooled individual patient-level data from 3 randomized clinical trials (ISAR-TEST 3, ISAR-TEST 4 and LEADERS) comparing biodegradable polymer DES with durable polymer SES. Clinical outcomes out to 4years were assessed. The primary end point was the composite of cardiac death, myocardial infarction and target-lesion revascularization. Secondary end points were target lesion revascularization and definite or probable stent thrombosis. RESULTS Of 1094 patients with diabetes included in the present analysis, 657 received biodegradable polymer DES and 437 durable polymer SES. At 4years, the incidence of the primary end point was similar with BP-DES versus SES (hazard ratio=0.95, 95% CI=0.74-1.21, P=0.67). Target lesion revascularization was also comparable between the groups (hazard ratio=0.89, 95% CI=0.65-1.22, P=0.47). Definite or probable stent thrombosis was significantly reduced among patients treated with BP-DES (hazard ratio=0.52, 95% CI=0.28-0.96, P=0.04), a difference driven by significantly lower stent thrombosis rates with BP-DES between 1 and 4years (hazard ratio=0.15, 95% CI=0.03-0.70, P=0.02). CONCLUSIONS In patients with diabetes, biodegradable polymer DES, compared to durable polymer SES, were associated with comparable overall clinical outcomes during follow-up to 4years. Rates of stent thrombosis were significantly lower with BP-DES.

Long-term outcomes of biodegradable versus durable polymer drug-eluting stents in patients with acute ST-segment elevation myocardial infarction: a pooled analysis of individual patient data from three randomised trials

Aims: Arterial plaque rupture and thrombus characterise ST-elevation myocardial infarction (STEMI) and may aggravate delayed arterial healing following durable polymer drug-eluting stent (DP-DES) implantation. Biodegradable polymer (BP) may improve biocompatibility. We compared long-term outcomes in STEMI patients receiving BP-DES vs. durable polymer sirolimus-eluting stents (DP-SES). Methods and results: We pooled individual patient-level data from three randomised clinical trials (ISAR-TEST-3, ISAR-TEST-4 and LEADERS) comparing outcomes from BP-DES with DP-SES at four years. The primary endpoint (MACE) comprised cardiac death, MI, or target lesion revascularisation (TLR). Secondary endpoints were TLR, cardiac death or MI, and definite or probable stent thrombosis. Of 497 patients with STEMI, 291 received BP-DES and 206 DP-SES. At four years, MACE was significantly reduced following treatment with BP-DES (hazard ratio [HR] 0.59, 95% CI: 0.39-0.90; p=0.01) driven by reduced TLR (HR 0.54, 95% CI: 0.30-0.98; p=0.04). Trends towards reduction were seen for cardiac death or MI (HR 0.63, 95% CI: 0.37-1.05; p=0.07) and definite or probable stent thrombosis (3.6% vs. 7.1%; HR 0.49, 95% CI: 0.22-1.11; p=0.09). Conclusions: In STEMI, BP-DES demonstrated superior clinical outcomes to DP-SES at four years. Trends towards reduced cardiac death or myocardial infarction and reduced stent thrombosis require corroboration in specifically powered trials.

Antibody-Drug Conjugates for Tumor Targeting-Novel Conjugation Chemistries and the Promise of non-IgG Binding Proteins

Antibody-drug conjugates (ADCs) have emerged as a promising class of anticancer agents, combining the specificity of antibodies for tumor targeting and the destructive potential of highly potent drugs as payload. An essential component of these immunoconjugates is a bifunctional linker capable of reacting with the antibody and the payload to assemble a functional entity. Linker design is fundamental, as it must provide high stability in the circulation to prevent premature drug release, but be capable of releasing the active drug inside the target cell upon receptor-mediated endocytosis. Although ADCs have demonstrated an increased therapeutic window, compared to conventional chemotherapy in recent clinical trials, therapeutic success rates are still far from optimal. To explore other regimes of half-life variation and drug conjugation stoichiometries, it is necessary to investigate additional binding proteins which offer access to a wide range of formats, all with molecularly defined drug conjugation. Here, we delineate recent progress with site-specific and biorthogonal conjugation chemistries, and discuss alternative, biophysically more stable protein scaffolds like Designed Ankyrin Repeat Proteins (DARPins), which may provide such additional engineering opportunities for drug conjugates with improved pharmacological performance.

The use of microviscometry to study polymer dissolution from solid dispersion drug delivery systems

Solid dispersions can be used to improve dissolution of poorly soluble drugs and PVP is a common polymeric carrier in such systems. The mechanisms controlling release of drug from solid dispersions are not fully understood and proposed theories are dependent on an understanding of the dissolution behaviour of both components of the dispersion. This study uses microviscometry to measure small changes in the viscosity of the dissolution medium as the polymer dissolves from ibuprofen-PVP solid dispersions. The microviscometer determines the dynamic and kinematic viscosity of liquids based on the rolling/falling ball principle. Using a standard USP dissolution apparatus, the dissolution of the polymer from the solid dispersion was easily measured alongside drug release. Drug release was found to closely follow polymer dissolution at the molecular weights and ratios used. The combination of sensitivity and ease of use make microviscometry a valuable technique for the elucidation of mechanisms governing drug release from polymeric delivery systems. © 2004 Elsevier B.V. All rights reserved.

Synthesis of triplex-forming oligonucleotide conjugates of the anticancer drug temodal

Covalent attachment of the anticancer drugs temozolomide (Temodal) and mitozolomide to triplex-forming oligonucleotides (TFOs) is a potential way of targeting these alkylating agents to specific gene sequences to maximise site-selectivity. In this work, polypyrimidine TFO conjugates of both drugs were synthesised and targeted to duplex DNA in an attempt to effect site-specific alkylation of guanine residues. Concurrently, in an attempt to enhance the triple helix stability of TFOs at neutral pH, the thermal stabilities of triplexes formed from TFOs containing isoguanine, 2-O-benzyl- and 2-O-allyl-adenine were evaluated. A novel cleavage and deprotection procedure was developed which allowed for the solid phase synthesis of the base-sensitive TFO-drug conjugates using a recently developed silyl-linked controlled pore glass (SLCPG) support. Covalent attachment of either temozolomide or mitozolomide at the 5'-end of TFO conjugates caused no destabilisation of the triplexes studied. The synthesis of a phosphoramidite derivative of mitozolomide enabled direct incorporation of this reagent into a model sequence during DNA synthesis. After cleavage and deprotection of the TFO-drug conjugate, the 5'-end mitozolomide residue was found to have decomposed presumably as a result of ring-opening of the tetrazinone ring. The base-sensitive antibacterial and antitumour agent, metronidazole, was also successfully incorporated at the 5'-end of the oligonucleotide d(T8) using conventional methods. Two C2-substituted derivatives of 2'-deoxyadenosine containing 2-O-benzyl and 2-O-allyl groups were synthesised. Hydrogenolysis of the 2-O-benzyl analogue provided a useful route, amenable to scale-up, for the synthesis of the rare nucleoside 2'-deoxyisoguanosine (isoG). Both the 2-O-allyl and 2-O-benzyl derivatives were incorporated into TFO sequences using phosphoramidite methodology. Thermal melting experiments showed that the 2-O-allyl and 2-O-benzyl groups caused marked destabilisation of the triple helices studied, in contrast to hexose-DNA duplexes, where aralkyl substituents caused significant stabilisation of duplexes. TFOs containing isoG were synthesised by Pd(O)-catalysed deallylation of 2-0-allyl adenine residues. These sequences containing isoG, in its N3- or 02-H tautomeric form, formed triple helices which were equally as stable as those containing adenine.

Investigating polymer-peptide conjugates and electrospinning for the production of advanced materials

This thesis describes the production of advanced materials comprising a wide array of polymer-based building blocks. These materials include bio-hybrid polymer-peptide conjugates, based on phenylalanine and poly(ethylene oxide), and polymers with intrinsic microporosity (PIMs). Polymer-peptides conjugates were previously synthesised using click chemistry. Due to the inherent disadvantages of the reported synthesis, a new, simpler, inexpensive protocol was sought. Three synthetic methods based on amidation chemistry were investigated for both oligopeptide and polymerpeptide coupling. The resulting conjugates produced were then assessed by various analytical techniques, and the new synthesis was compared with the established protocol. An investigation was also carried out focussing on polymer-peptide coupling via ester chemistry, involving deprotection of the carboxyl terminus of the peptide. Polymer-peptide conjugates were also assessed for their propensity to self-assemble into thixotropic gels in an array of solvent mixtures. Determination of the rules governing this particular self-assembly (gelation) was required. Initial work suggested that at least four phenylalanine peptide units were necessary for self-assembly, due to favourable hydrogen bond interactions. Quantitative analysis was carried out using three analytical techniques (namely rheology, FTIR, and confocal microscopy) to probe the microstructure of the material and provided further information on the conditions for self-assembly. Several polymers were electrospun in order to produce nanofibres. These included novel materials such as PIMs and the aforementioned bio-hybrid conjugates. An investigation of the parameters governing successful fibre production was carried out for PIMs, polymer-peptide conjugates, and for nanoparticle cages coupled to a polymer scaffold. SEM analysis was carried out on all material produced during these electrospinning experiments.