PEGylated Purpurin 18 with Improved Solubility: Potent Compounds for Photodynamic Therapy of Cancer.

Purpurin 18 derivatives with a polyethylene glycol (PEG) linker were synthesized as novel photosensitizers (PSs) with the goal of using them in photodynamic therapy (PDT) for cancer. These compounds, derived from a second-generation PS, exhibit absorption at long wavelengths; considerable singlet oxygen generation and, in contrast to purpurin 18, have higher hydrophilicity due to decreased logP. Together, these properties make them potentially ideal PSs. To verify this, we screened the developed compounds for cell uptake, intracellular localization, antitumor activity and induced cell death type. All of the tested compounds were taken up into cancer cells of various origin and localized in organelles known to be important PDT targets, specifically, mitochondria and the endoplasmic reticulum. The incorporation of a zinc ion and PEGylation significantly enhanced the photosensitizing efficacy, decreasing IC50 (half maximal inhibitory compound concentration) in HeLa cells by up to 170 times compared with the parental purpurin 18. At effective PDT concentrations, the predominant type of induced cell death was apoptosis. Overall, our results show that the PEGylated derivatives presented have significant potential as novel PSs with substantially augmented phototoxicity for application in the PDT of cervical, prostate, pancreatic and breast cancer.

Stanazolol derived ELISA as a sensitive forensic tool for the detection of multiple 17α-methylated anabolics.

Two valuable forensic tools based on enzyme-linked immunoassays (ELISAs) for the analysis of 17α-methylated steroids were developed using haptens of stanazolol and its conjugates with biotin. Haptens containing terminal carboxylic group were conjugated to bovine serum albumin (BSA), rabbit serum albumin (RSA) or ovalbumin (OVA). Eight batches of antisera (RAbs) obtained by immunization of rabbits were tested in an indirect competitive ELISA system using immobilization of RSA conjugate (RSA/hapten) and competitor immobilization of the biotinylated conjugate (AB-ELISA) to avidin (avidin/hapten). The best results were achieved with the RAb 212 antibodies in RSA/ST-3 and avidin/ST-10 assembled variants. For the RSA/ST-3 system, an IC50 of 0.3 ng/mL and a detection limit of 0.02 ng/mL were measured. In case of avidin/ST-10 variant, IC50 was of 3.9 ng/mL and a detection limit of 0.57 ng/mL were obtained. The effect of solvent was tested as well as the stability of coated microtiter plates over four-month period. The cross-reactivity of the developed assays with other anabolic steroids was tested and high sensitivity towards 17α-methylated steroids was observed. RSA/ST-3 assay showed significant cross-reactivity with 17α-methyltestosterone (81.2%), oxymetholone (30.4%), methandienone (10.0%) and methyl dihydrotestosterone (7.7%). Similarly, in the avidin/ST-10 assay, 17α-methyltestosterone (34.5%), mestanolone (32.1%), oxymetholone (22.7%), methandienone (14.2%), 9-dehydromethyltestosterone (12.5%) and oxandrolone (1.2%) exhibited high cross-reactivity. The functionality of the developed systems was verified by the successful identification of a series of 17α-methylated anabolic steroids in a set of real samples including pharmaceutical preparations seized by the Police of the Czech Republic on the black market.

Formation of 5α-Sitostan-3-one, 5α-Campestan-3-one, and Steroidal Hydrocarbons in Edible Oils during Catalytic Hydrogenation.

Targeted analysis confirmed the presence of new phytosterol degradation products in fully hydrogenated commercial samples. EI-MS, APCI-MS, and 1D-NMR experiments led to the identification of 10 novel markers of catalytic hydrogenation, among which 5α-sitostan-3-one and 5α-campestan-3-one, isomers of saturated and monounsaturated steroidal hydrocarbons, were reported in edible oils for the first time. Examination of the phytosterol degradation mechanism was done by the catalytic transfer deuteration technique. The mitigation strategy of potentially detrimental compounds included optimization of processing parameters. The effect of catalyst dosage (≤0.1% based on Ni basis) and temperature region (>180 °C) were the most crucial factors in phytosterol degradation control.

Oxime-based 19-nortestosterone-pheophorbide a conjugate: bimodal controlled release concept for PDT.

Photodynamic therapy has become a feasible direction for the treatment of both malignant and non-malignant diseases. It has been in the spotlight since FDA regulatory approval was granted to several photosensitizers worldwide. Nevertheless, there are still strong limitations in the targeting specificity that is vital to prevent systemic toxicity. Here, we report the synthesis and biological evaluation of a novel bimodal oxime conjugate composed of a photosensitizing drug, red-emitting pheophorbide a, and nandrolone (NT), a steroid specifically binding the androgen receptor (AR) commonly overexpressed in various tumors. We characterized the physico-chemical properties of the NT-pheophorbide a conjugate (NT-Pba) and singlet oxygen generation. Because light-triggered therapies have the potential to provide important advances in the treatment of hormone-sensitive cancer, the biological potential of this novel specifically-targeted photosensitizer was assessed in prostatic cancer cell lines in vitro using an AR-positive (LNCaP) and an AR-negative/positive cell line (PC-3). U-2 OS cells, both with and without stable AR expression, were used as a second cell line model. Interestingly, we found that the NT-Pba conjugate was not only photodynamically active and AR-specific, but also that its phototoxic effect was more pronounced compared to pristine pheophorbide a. We also examined the intracellular localization of NT-Pba. Live-cell fluorescence microscopy provided clear evidence that the NT-Pba conjugate localized in the endoplasmic reticulum and mitochondria. Moreover, we performed a competitive localization study with the excess of nonfluorescent NT, which was able to displace fluorescent NT-Pba from the cell interior, thereby further confirming the binding specificity. The oxime ether bond degradation was assayed in living cells by both real-time microscopy and a steroid receptor reporter assay using AR U-2 OS cells. Thus, NT-Pba is a promising candidate for both the selective targeting and eradication of AR-positive malignant cells by photodynamic therapy.

Selectivity of original C-hexopyranosyl calix[4]arene conjugates towards lectins of different origin.

As a part of ongoing activities towards the design of ligands against pathogenic lectins, a synthesis of original α-C-galacto/α-C-manno/α-C-fucopyranosyl glycomimetics based on a calix[4]arene scaffold and their binding evaluation is described. The interactions of the glycomimetics with seven lectins of various origins were carried out using agglutination inhibition assays. The 1,3-alternate tetra-C-fucosylated ligand and its derivative having a tertBu group at the upper rim of the calix[4]arene scaffold were the most potent towards the AAL lectin family (RSL, AFL, AAL, AOL) and BC2L-C. As AFL and RSL originate from important human (Aspergillus fumigatus) and plant (Ralstonia solanacearum) pathogens, the inhibition potency of both leading structures was assessed by surface plasmon resonance. With AFL, both structures exhibited an approximately three orders of magnitude increase in affinity compared to the reference l-fucose. The role of tertBu groups as "aglycon-assisted" events was illustrated by NMR. Furthermore, both compounds showed significantly increased ability to inhibit BC2L-C (from human pathogen Burkholderia cenocepacia) cell agglutination and were able to cross-link whole B. cenocepacia cells. Although the ligands failed to significantly inhibit the agglutination activity of LecA and LecB from Pseudomonas aeruginosa, tetra-C-galactosylated calix[4]arene with tertBu groups at the upper rim of the 1,3-alternate conformation inhibited P. aeruginosa biofilm formation efficiently. This systematic and comprehensive study highlights the fact that hydrolytically stable polyvalent C-glycomimetics should be regarded as potent and selective ligands capable of acting as antiadhesive agents.

Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders.

The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analysed in detail and showed an intensive labelling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 signal in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.

Immunoassay for determination of trilobolide.

Trilobolide (Tb) is a pharmacologically interesting sesquiterpene lactone isolated from Laser trilobum (L.) Borkh. Structural relation to a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin bring promising prospects for Tb to be used in the development of new anti-cancer drugs. As long as there are still unanswered questions regarding its investigation, a need for novel analytical tools emerge. Since immunoassays serve as one of powerful tools within the investigation of natural products, the development of indirect competitive enzyme-linked immunosorbent assay (ELISA) utilizing coating based on avidin-biotin technology is described. In our set-up of ELISA, newly synthesized biotinylated Tb served as immobilized competitor. Tb-carboxymethyloxime-bovine serum albumin (BSA) and Tb-succinoyl-BSA conjugates were used separately for immunization of rabbits. Two sets of polyclonal antibodies (RAbs) were obtained. Antibodies against Tb-succinoyl-BSA conjugate (RAb No. 206) were chosen as the best. Under optimized conditions, limit of detection and 50% intercept of our ELISA were 849pg/mL and 8.89ng/mL, respectively. The cross-reactivity (CR) was tested on 10 structurally related compounds and CR did not exceed 6.1%. The reproducibility of the system is expressed as intra- and inter-assay coefficients of variation (9.7% and 11.4%, respectively). Based on conducted experiments, we proposed the use of ELISA for quantification of Tb in complex biological matrices such as plant extracts. A method was applied to analyze three extracts obtained from different parts of L. trilobum. Data obtained were compared to those acquired by UHPLC-MS/MS. The concordance between the methods (103-87%) showed the ability of ELISA to quantify Tb.