Structure-guided optimization of 3-hydroxybenzoisoxazole derivatives as inhibitors of Aldo-keto reductase 1C3 (AKR1C3) to target prostate cancer.

AKR1C3 is an enzyme that is overexpressed in several types of radiotherapy- and chemotherapy-resistant cancers. Despite AKR1C3 is a validated target for drug development, no inhibitor has been approved for clinical use. In this manuscript, we describe our study of a new series of potent AKR1C3-targeting 3-hydroxybenzoisoxazole based inhibitors that display high selectivity over the AKR1C2 isoform and low micromolar activity in inhibiting 22Rv1 prostate cancer cell proliferation. In silico studies suggested proper substituents to increase compound potency and provided with a mechanistic explanation that could clarify their different activity, later confirmed by X-ray crystallography. Both the in-silico studies and the crystallographic data highlight the importance of 90° rotation around the single bond of the biphenyl group, in ensuring that the inhibitor can adopt the optimal binding mode within the active pocket. The p-biphenyls that bear the meta-methoxy, and the ortho- and meta-trifluoromethyl substituents (in compounds 6a, 6e and 6f respectively) proved to be the best contributors to cellular potency as they provided the best IC50 values in series (2.3, 2.0 and 2.4 µM respectively) and showed no toxicity towards human MRC-5 cells. Co-treatment with scalar dilutions of either compound 6 or 6e and the clinically used drug abiraterone led to a significant reduction in cell proliferation, and thus confirmed that treatment with both CYP171A1-and AKR1C3-targeting compounds possess the potential to intervene in key steps in the steroidogenic pathway. Taken together, the novel compounds display desirable biochemical potency and cellular target inhibition as well as good in-vitro ADME properties, which highlight their potential for further preclinical studies.

New aldo-keto reductase 1C3 (AKR1C3) inhibitors based on the hydroxytriazole scaffold.

The aldo-keto reductase 1C3 (AKR1C3) enzyme is considered an attractive target in Castration Resistant Prostate Cancer (CRPC) because of its role in the biosynthesis of androgens. Flufenamic acid, a non-selective AKR1C3 inhibitor, has previously been subjected to bioisosteric modulation to give rise to a series of compounds with the hydroxytriazole core. In this work, the hit compound of the previous series has been modulated further, and new, more potent, and selective derivatives have been obtained. The poor solubility of the most active compound (cpd 5) has been improved by substituting the triazole core with an isoxazole heteronucleous, with similar enzymatic activity being retained. Potent AKR1C3 inhibition is translated into antiproliferative effects against the 22RV1 CRPC cellular model, and the in-silico design, synthesis and biological activity of new compounds are described herein. Compounds have also been assayed in combination with two approved antitumor drugs, abiraterone and enzalutamide.

Effects of the Molecular Weight of Hyaluronic Acid in a Carbon Nanotube Drug Delivery Conjugate.

Hyaluronic acid (HA) is a ubiquitous biopolymer involved in many pathophysiological roles. One HA receptor, the cluster of differentiation CD44 protein, is often overexpressed in tumor cells. As such, HA has attracted considerable interest in the development of drug delivery formulations, given its intrinsic targetability toward CD44 overexpressing cells. The present study is focused on examining the correlation of HA molecular weight with its targetability properties. A library of conjugates obtained by linking the amino group of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) to the carboxylic residues of HA of different molecular weight (6.4, 17, 51, 200, and 1,500 kDa) were synthesized and fully characterized. The HA-DMPE conjugates were then used to non-covalently functionalize the highly hydrophobic single-walled carbon nanotubes (CNT), and further encapsulate the anticancer drug doxorubicin (DOX). Our results show that the complexes DOX/CNT/HA-DMPE maintain very good and stable dispersibility. Drug release studies indicated a pH-responsive release of the drug from the nanocarrier. Cell viability tests demonstrated that all HA modified CNTs have good biocompatibility, and specific targeting toward cells overexpressing the CD44 receptor. Among all the molecular weights tested, the 200 kDa HA showed the highest increase in cellular uptake and cytotoxic activity. All these promising attributes make CNT/HA200-DMPE a "smart" platform for tumor-targeted delivery of anticancer agents.

SWCNT-porphyrin nano-hybrids selectively activated by ultrasound: an interesting model for sonodynamic applications.

Sonodynamic therapy (SDT) is an innovative anticancer approach, based on the excitation of a given molecule (usually a porphyrin) by inertial acoustic cavitation that leads to cell death via the production of reactive oxygen species (ROS). This study aims to prepare and characterize nanosystems based on porphyrin grafted carbon nanotubes (SWCNTs), to understand some aspects of the mechanisms behind the SDT phenomenon. Three different porphyrins have been covalently linked to SWCNTs using either Diels-Alder or 1,3-dipolar cycloadditions. ROS production and cell viability have been evaluated upon ultrasound irradiation. Despite the low porphyrin content linked on the SWCNT, these systems have shown high ROS production and high tumour-cell-killing ability. The existence of a PET (photoinduced electron transfer)-like process would appear to be able to explain these observations. Moreover, the demonstrated ability to absorb light limits the impact of side effects due to light-excitation.

Pentamidine-Loaded Lipid and Polymer Nanocarriers as Tunable Anticancer Drug Delivery Systems.

Initially developed as a synthetic analogue of insulin, pentamidine (PTM) is an antimicrobial drug that has recently shown in vitro and in vivo anticancer activity. Nevertheless, systemic administration of PTM causes severe side effects, especially nephrotoxicity. Here we propose the association of PTM to different biocompatible nanosystems in order to compare the physicochemical characteristics of the loaded nanocarriers and their influence on the drug cytotoxicity toward cancer cells. In particular, PTM (as free base or with different counterions) was encapsulated into liposomes and poly(lactide-co-glycolide) (PLGA) nanoparticles and all the formulations have been deeply characterized concerning mean diameter, polydispersity index, zeta potential, stability, morphology, PTM loading, and drug release profile. The anticancer activity was evaluated on a human ovarian cancer cell line over 72 h. Results showed that PTM is efficiently loaded into liposomes with a transmembrane citrate or sulfate gradient; concerning PLGA nanoparticles, important association occurred, thanks to ionic interactions between the drug and the polymer. The in vitro studies confirmed the anticancer activity of PTM, which was gradually released with different profiles depending on the drug form and the nanocarrier structure.

Bioisosteres of Indomethacin as Inhibitors of Aldo-Keto Reductase 1C3.

Aldo-keto reductase 1C3 (AKR1C3) is an attractive target in drug design for its role in resistance to anticancer therapy. Several nonsteroidal anti-inflammatory drugs such as indomethacin are known to inhibit AKR1C3 in a nonselective manner because of COX-off target effects. Here we designed two indomethacin analogues by proposing a bioisosteric connection between the indomethacin carboxylic acid function and either hydroxyfurazan or hydroxy triazole rings. Both compounds were found to target AKR1C3 in a selective manner. In particular, hydroxyfurazan derivative is highly selective for AKR1C3 over the 1C2 isoform (up to 90-times more) and inactive on COX enzymes. High-resolution crystal structure of its complex with AKR1C3 shed light onto the binding mode of the new inhibitors. In cell-based assays (on colorectal and prostate cancer cells), the two indomethacin analogues showed higher potency than indomethacin. Therefore, these two AKR1C3 inhibitors can be used to provide further insight into the role of AKR1C3 in cancer.

Potent and selective aldo-keto reductase 1C3 (AKR1C3) inhibitors based on the benzoisoxazole moiety: application of a bioisosteric scaffold hopping approach to flufenamic acid.

The aldo-keto reductase 1C3 (AKR1C3) isoform plays a vital role in the biosynthesis of androgens and is considered an attractive target in prostate cancer (PCa). No AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid and indomethacine are non-steroidal anti-inflammatory drugs known to inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. Recently, we employed a scaffold hopping approach to design a new class of potent and selective AKR1C3 inhibitors based on a N-substituted hydroxylated triazole pharmacophore. Following a similar strategy, we designed a new series focused around an acidic hydroxybenzoisoxazole moiety, which was rationalised to mimic the benzoic acid role in the flufenamic scaffold. Through iterative rounds of drug design, synthesis and biological evaluation, several compounds were discovered to target AKR1C3 in a selective manner. The most promising compound of series (6) was found to be highly selective (up to 450-fold) for AKR1C3 over the 1C2 isoform with minimal COX1 and COX2 off-target effects. Other inhibitors were obtained modulating the best example of hydroxylated triazoles we previously presented. In cell-based assays, the most promising compounds of both series reduced the cell proliferation, prostate specific antigen (PSA) and testosterone production in AKR1C3-expressing 22RV1 prostate cancer cells and showed synergistic effect when assayed in combination with abiraterone and enzalutamide. Structure determination of AKR1C3 co-crystallized with one representative compound from each of the two series clearly identified both compounds in the androstenedione binding site, hence supporting the biochemical data.

Hyaluronated mesoporous silica nanoparticles for active targeting: influence of conjugation method and hyaluronic acid molecular weight on the nanovector properties.

We have prepared and evaluated the physico-chemical and biological properties of four different hyaluronated mesoporous silica nanoparticles (MSNs) samples (MSN/HA). Hyaluronic acid (HA) with two different molecular weights (200 and 6.4 kDa) was used for the conjugation of aminopropyl-functionalized MSN (NH2-MSN), following two different procedures. Namely, samples HA200A and HA6.4A were prepared by reacting activated HA with NH2-MSN (method A), while samples HA200B and HA6.4B were obtained carrying out HA activation in the presence of the nanoparticles (method B). The four samples showed similar hydrophilicity, but clear differences in the HA loading, textural properties, surface charge and stability of the suspensions. More in detail, conjugation using low molecular weight HA with method A resulted in low HA loading, with consequent scarce effects on dispersity and stability in physiological media. The highest yield and corresponding best performances were obtained with method B using high molecular weight HA. HA loading and molecular weight also influenced in a concerted way the biological response towards the MSNs of CD44 target cancer cells (CD44+) and control cells (CD44-): MDA-MB-231 and A2780, respectively. The absence of cytotoxicity was assessed. Moreover, the targeting ability of the best performing MSN/HA was confirmed by cellular uptake studies.

Hydroxytriazole derivatives as potent and selective aldo-keto reductase 1C3 (AKR1C3) inhibitors discovered by bioisosteric scaffold hopping approach.

The aldo-keto reductase 1C3 isoform (AKR1C3) plays a vital role in the biosynthesis of androgens, making this enzyme an attractive target for castration-resistant prostate cancer therapy. Although AKR1C3 is a promising drug target, no AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid, a non-steroidal anti-inflammatory drug, is known to potently inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. To diminish off-target effects, we have applied a scaffold hopping strategy replacing the benzoic acid moiety of flufenamic acid with an acidic hydroxyazolecarbonylic scaffold. In particular, differently N-substituted hydroxylated triazoles were designed to simultaneously interact with both subpockets 1 and 2 in the active site of AKR1C3, larger for AKR1C3 than other AKR1Cs isoforms. Through computational design and iterative rounds of synthesis and biological evaluation, novel compounds are reported, sharing high selectivity (up to 230-fold) for AKR1C3 over 1C2 isoform and minimal COX1 and COX2 off-target inhibition. A docking study of compound 8, the most interesting compound of the series, suggested that its methoxybenzyl substitution has the ability to fit inside subpocket 2, being involved in π-π staking interaction with Trp227 (partial overlapping) and in a T-shape π-π staking with Trp86. This compound was also shown to diminish testosterone production in the AKR1C3-expressing 22RV1 prostate cancer cell line while synergistic effect was observed when 8 was administered in combination with abiraterone or enzalutamide.

Mesoporous silica nanoparticles as a promising skin delivery system for methotrexate.

The systemic administration of methotrexate (MTX), a commonly used, antineoplastic drug which is also used in cutaneous disorders, is primarily associated with prolonged retention in the body and consequently with side effects. Innovative drug delivery techniques and alternative administration routes would therefore contribute to its safe and effective use. The general objective of this study is thus the development of MTX-based preparations for the topical treatment of skin disorders. MCM-41-like nanoparticles (MSN), are herein proposed as carriers which can improve the cutaneous absorption and hence the bioavailability and efficacy of MTX. The MTX/MSN complex, prepared via the impregnation procedure, has been physico-chemically characterized, while its cell cultures have had their biocompatibility and bioactivity tested. Furthermore, a series of stable MTX-based dermal formulations has been developed, some containing shea butter, a natural fat. Ex-vivo porcine skin absorption and the transepidermal permeation of MTX have also been monitored in a variety of media using Franz diffusion cells. Interestingly, the epidermal accumulation of the active molecule was increased by its inclusion into MSN, regardless of the surrounding medium. Furthermore, the presence of shea butter enhanced the skin uptake of the drug both in the free and in the loaded form.

Delivery of Gemcitabine Prodrugs Employing Mesoporous Silica Nanoparticles.

In this paper, mesoporous silica nanoparticles (MSNs) were studied as vehicles for the delivery of the antitumoral drug gemcitabine (GEM) and of its 4-(N)-acyl derivatives, (4-(N)-valeroyl-(C5GEM), 4-(N)-lauroyl-(C12GEM) and 4-(N)-stearoyl-gemcitabine (C18GEM)). The loading of the GEM lipophilic prodrugs on MSNs was explored with the aim to obtain both a physical and a chemical protection of GEM from rapid plasmatic metabolization. For this purpose, MSNs as such or with grafted aminopropyl and carboxyethyl groups were prepared and characterized. Then, their different drug loading capacity in relation to the nature of the functional group was evaluated. In our experimental conditions, GEM was not loaded in any MSNs, while C12GEM was the most efficiently encapsulated and employed for further evaluation. The results showed that loading capacity increased with the presence of functional groups on the nanoparticles; similarly, the presence of functional groups on MSNs' surface influenced the drug release profile. Finally, the cytotoxicity of the different preparations was evaluated and data showed that C12GEM loaded MSNs are less cytotoxic than the free drug with an activity that increased with the incubating time, indicating that all these systems are able to release the drug in a controlled manner. Altogether, the results demonstrate that these MSNs could be an interesting system for the delivery of anticancer drugs.

Mesoporous silica as topical nanocarriers for quercetin: characterization and in vitro studies.

The flavonoid quercetin is extensively studied for its antioxidant and chemopreventive properties. However the poor water-solubility, low stability and short half-life could restrict its use in skin care products and therapy. The present study was aimed to evaluate the potential of aminopropyl functionalized mesoporous silica nanoparticles (NH2-MSN) as topical carrier system for quercetin delivery. Thermo gravimetric analysis, X-ray diffraction, high resolution transmission electron microscopy, nitrogen adsorption isotherms, FT-IR spectroscopy, zeta potential measurements and differential scanning calorimetry allowed analyzing with great detail the organic-inorganic molecular interaction. The protective effect of this vehicle on UV-induced degradation of the flavonoid was investigated revealing a certain positive influence of the inclusion on the photostability over time. Epidermal accumulation and transdermal permeation of this molecule were ex vivo evaluated using porcine skin mounted on Franz diffusion cells. The inclusion complexation with the inorganic nanoparticles increased the penetration of quercetin into the skin after 24h post-application without transdermal delivery. The effect of quercetin alone or given as complex with NH2-MSN on proliferation of JR8 human melanoma cells was evaluated by sulforhodamine B colorimetric proliferation assay. At a concentration 60 µM the complex with NH2-MSN was more effective than quercetin alone, causing about 50% inhibition of cell proliferation.

Preparation and characterization of novel poly(ethylene glycol) paclitaxel derivatives.

Paclitaxel has been found to be very effective against several human cancers; one of the major problems with its use is its poor solubility, which makes necessary its solubilization with excipients that can determine allergic reactions often severe. The aim of this study is to develop highly water-soluble prodrugs of paclitaxel. For this purpose we prepared a series of new paclitaxel-poly(ethylene glycol) (PEG) conjugates that were characterized and evaluated for their in vitro stability and cytotoxicity. In particular, in order to modulate the release of paclitaxel from prodrugs, we prepared different compounds introducing PEG in the drug C2' and/or C7 positions via ester or carbamate linkage. The conjugates were obtained in high purity and good yield. The carbamate prodrugs were highly stable in different media, while the compounds obtained linking PEG at C2' position through an ester bond showed lower stability. Finally, the cytotoxic activity of the conjugates was evaluated on two cancer cell lines and the results showed that all the derivatives had a reduced cytotoxicity compared to that of paclitaxel.

Ruthenium polypyridyl squalene derivative: a novel self-assembling lipophilic probe for cellular imaging.

Transition metal complexes provide a promising avenue for designing new therapeutic and diagnostic agents. In particular, ruthenium(II) polypyridyl complexes are useful for studying cellular uptake, due to their easy synthesis and unique photophysical properties. Dyes are frequently combined with material substrates to modulate their properties, enhance stability, reduce toxicity, and improve delivery. A novel Ru polypyridyl complex linked to a derivative of the natural lipid squalene (Ru-BIPPBI-hx-SQ) is described. Using the solvent displacement method, Ru-BIPPBI-hx-SQ easily self-assembles into nanosized aggregates in aqueous solution, as characterized by dynamic light scattering. The nanoassemblies exhibit long-lived and intense luminescence. Preliminary biological assessment showed them to be non-toxic; they are efficiently and rapidly transported across the cell membrane without requiring its permeabilization. Ru-labeled nanoassemblies are likely to be significant cellular-imaging tools, probing cellular events at very low concentrations. Moreover co-nanoassembly, with drug-derivatives based on squalenoylation technology, including gemcitabine and paclitaxel, has given interesting preliminary results.