Search

Herein we describe the design and biological evaluation of a novel antitumor therapeutic platform that combines the most favorable properties of small-molecule drug conjugates (SMDCs) and antibody drug conjugates (ADCs). Although the small size of SMDCs, compared to ADCs, is an appealing feature for their application in the treatment of solid tumors, SMDCs usually suffer from poor pharmacokinetics, which severely limits their therapeutic efficacy. To overcome this limitation, in this proof-of-concept study we grafted an α-amanitin-based SMDC that targets prostate cancer cells onto an immunoglobulin Fc domain via a two-step "program and arm" chemoenzymatic strategy. We demonstrated the superior pharmacokinetic properties and therapeutic efficacy of the resulting Fc-SMDC over the SMDC in a prostate cancer xenograft mouse model. This approach may provide a general strategy toward effective antitumor therapeutics combining small size with pharmacokinetic properties close to those of an ADC.

RGD-α-amanitin and isoDGR-α-amanitin conjugates were synthesized by joining integrin ligands to α-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified α V β 3 receptor, retaining good binding affinity, in the same nanomolar range as the free ligands. The antiproliferative activity of the conjugates was evaluated in three cell lines possessing different levels of α V β 3 integrin expression: human glioblastoma U87 (α V β 3 +), human lung carcinoma A549 (α V β 3 -) and breast adenocarcinoma MDA-MB-468 (α V β 3 -). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-α-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than α-amanitin. Apparently, for all these α-amanitin conjugates there is no correlation between the cytotoxicity and the expression of α V β 3 integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-α-amanitin conjugates is governed by an integrin-mediated binding and internalization process, competition experiments were carried out in which the conjugates were tested with U87 (α V β 3 +, α V β 5 +, α V β 6 -, α 5 β 1 +) and MDA-MB-468 (α V β 3 -, α V β 5 +, α V β 6 +, α 5 β 1 -) cells in the presence of excess cilengitide, with the aim of blocking integrins on the cell surface. Using the MDA-MB-468 cell line, a fivefold increase of the IC 50 was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only α V β 3 , but also α V β 5 , α V β 6 , and α 5 β 1 . These data indicate that in this case the cyclo[DKP-isoDGR]-α-amanitin conjugates are possibly internalized by a process mediated by integrins different from α V β 3 (e.g., α V β 5 ).

Gastric cancer (GC) is the 3rd deadliest cancer worldwide, due to limited treatment options and late diagnosis. Human epidermal growth factor receptor-2 (HER2) is overexpressed in ∼20% of GC cases and anti-HER2 antibody trastuzumab in combination with conventional chemotherapy, is recognized as standard therapy for HER2-positive metastatic GC. This strategy improves GC patients' survival by 2-3 months, however its optimal results in breast cancer indicate that GC survival may be improved. A new photoimmunoconjugate was developed by conjugating a porphyrin with trastuzumab (Trast:Porph) for targeted photodynamic therapy in HER2-positive GC. Using mass spectrometry analysis, the lysine residues in the trastuzumab structure most prone for porphyrin conjugation were mapped. The in vitro data demonstrates that Trast:Porph specifically binds to HER2-positive cells, accumulates intracellularly, co-localizes with lysosomal marker LAMP1, and induces massive HER2-positive cell death upon cellular irradiation. The high selectivity and cytotoxicity of Trast:Porph based photoimmunotherapy is confirmed in vivo in comparison with trastuzumab alone, using nude mice xenografted with a HER2-positive GC cell line. In the setting of human disease, these data suggest that repetitive cycles of Trast:Porph photoimmunotherapy may be used as an improved treatment strategy in HER2-positive GC patients., (© 2017 UICC.)

5,10,15,20-tetrakis(1-methylpyridinium-4-yl)-porphyrin tetra-iodide (TMPyP), a potent water-soluble photosensitizer (PS) used in antimicrobial applications, was encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (TMPyP-PLGA) for topical delivery purposes. Nanoparticles resulted in a mean particle size around 130nm, narrow polydispersity index (PdI), spherical morphology and association efficiency up to 93%. Free TMPyP and TMPyP-PLGA nanoparticles were incorporated into Carbopol(®) hydrogels, resulting in controlled TMPyP release of about 60% and 20% after 4.5h, respectively. Critical properties such as appearance, clarity, viscosity and pH were maintained over time, as hydrogels were stable during 6 months at 4°C, 25°C/60% RH and 40°C/75% RH. For photodynamic applications, the photoproduction of singlet oxygen from these hydrogels was quite efficient being both formulations very photostable after 20min. No TMPyP permeation through pig ear skin was observed after 24h, and histological assays did not show relevant damages in surrounding tissues. All these excellent characteristics make them promising platforms for photodynamic applications through topical clinical use., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The current therapeutic strategies are based on the design of multifunctional drug candidates able to interact with various disease related targets. Drugs that have the ability to scavenge reactive oxygen species (ROS), beyond their main therapeutic action, may prevent the oxidative damage of biomolecules. Therefore, analytical approaches that monitor in a continuous mode the ability of drugs to counteract peroxidation of physiologically relevant biotargets are required. In the present work, a microplate spectrophotometric assay is proposed to evaluate the ability of selected cardiovascular drugs, including angiotensin-converting enzyme (ACE) inhibitors, β -blockers and statins to prevent protein peroxidation. Myoglobin, which is a heme protein, and peroxyl radicals generated from thermolysis of 2,2'-azo-bis(2-amidinopropane) dihydrochloride at 37 °C, pH 7.4 were selected as protein model and oxidative species, respectively. Myoglobin peroxidation was continuously monitored by the absorbance decrease at 409 nm and the ability of drugs to counteract protein oxidation was determined by the calculation of the area under the curve upon the myoglobin oxidation. Fluvastatin (AUC₅₀=12.5 ± 1.2 μM) and enalapril (AUC₅₀=15.2 ± 1.8 μM) showed high ability to prevent myoglobin peroxidation, providing even better efficiency than endogenous antioxidants such as reduced glutathione. Moreover, labetalol, enalapril and fluvastatin prevent the autoxidation of myoglobin, while glutathione showed a pro-oxidant effect., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Targeting photosensitizers to cancer cells by conjugating them with specific antibodies, able to recognize and bind to tumor-associated antigens, is today one of the most attractive strategies in photodynamic therapy (PDT). This comprehensive review updates on chemical routes available for the preparation of photo-immunoconjugates (PICs), which show dual chemical and biological functionalities: photo-properties of the photosensitizer and the immunoreactivity of the antibody. Moreover, photobiological results obtained with such photo-immunoconjugates using in vitro and in vivo cancer models are also discussed.