Drug Repositioning for Ovarian Cancer Treatment: An Update.

Ovarian cancer (OC) is one of the most prevalent malignancies in female reproductive organs, and its 5-year survival is below 45%. Despite the advances in surgical and chemotherapeutic options, OC treatment is still a challenge, and new anticancer agents are urgently needed. Drug repositioning has gained significant attention in drug discovery, representing a smart way to identify new clinical applications for drugs whose human safety and pharmacokinetics have already been established, with great time and cost savings in pharmaceutical development endeavors. This review offers an update on the most promising drugs repurposable for OC treatment and/or prevention.

Terpyridine Functionalized Cyclodextrin Nanoparticles as Carriers for Doxorubicin: Analysis of In Vitro Antiproliferative Activity.

BACKGROUND/AIM: We have recently described the development of cyclodextrin-based nanoparticles (NPs) functionalized with terpyridine and decorated with biotin-terpyridine ligands via Cu(II) and Fe(II) coordination. In the present study, we report the performance of these novel NPs as a delivery system for anticancer drugs. In particular, we analyzed the feasibility of loading these new NPs with the topoisomerase II inhibitor Doxorubicin (Doxo), still administered to patients to treat different forms of cancers. We developed Doxo-encapsulated polymeric NPS to generate nanoformulations with higher efficacy than free Doxo. MATERIALS AND METHODS: We investigated the inhibition of cell proliferation in A2780, A549, SKHep1, and MDA-MB-453 cancer cell lines using the MTT assay. RESULTS: NPs loaded with Doxo displayed higher antiproliferative activity than free Doxo. CONCLUSION: The NPs generated in this study inhibited the proliferation of cancer cells and were able to entrap the classic anticancer drug Doxo. The Doxo-loaded NP showed increased cytotoxicity in comparison to free Doxo.

Optimized spirooxindole-pyrazole hybrids targeting the p53-MDM2 interplay induce apoptosis and synergize with doxorubicin in A549 cells.

Recently, cancer research protocols have introduced clinical-stage spirooxindole-based MDM2 inhibitors. However, several studies reported tumor resistance to the treatment. This directed efforts to invest in designing various combinatorial libraries of spirooxindoles. Herein, we introduce new series of spirooxindoles via hybridization of the chemically stable core spiro[3H-indole-3,2'-pyrrolidin]-2(1H)-one and the pyrazole motif inspired by lead pyrazole-based p53 activators, the MDM2 inhibitor BI-0252 and promising molecules previously reported by our group. Single crystal X-ray diffraction analysis confirmed the chemical identity of a representative derivative. Fifteen derivatives were screened for cytotoxic activities via MTT assay against a panel of four cancer cell lines expressing wild-type p53 (A2780, A549, HepG2) and mutant p53 (MDA-MB-453). The hits were 8h against A2780 (IC50 = 10.3 µM) and HepG2 (IC50 = 18.6 µM), 8m against A549 (IC50 = 17.7 µM), and 8k against MDA-MB-453 (IC50 = 21.4 µM). Further MTT experiments showed that 8h and 8j potentiated doxorubicin activity and reduced its IC50 by at least 25% in combinations. Western blot analysis demonstrated that 8k and 8m downmodulated MDM2 in A549 cells. Their possible binding mode with MDM2 were simulated by docking analysis.

Terpyridine Glycoconjugates and Their Metal Complexes: Antiproliferative Activity and Proteasome Inhibition.

Metal terpyridine complexes have gained substantial interest in many application fields, such as catalysis and supramolecular chemistry. In recent years, the biological activity of terpyridine and its metal complexes has aroused considerable regard. On this basis, we synthesised new terpyridine derivatives of trehalose and glucose to improve the water solubility of terpyridine ligands and target them in cancer cells through glucose transporters. Glucose derivative and its copper(II) and iron(II) complexes showed antiproliferative activity. Interestingly, trehalose residue reduced the cytotoxicity of terpyridine. Moreover, we tested the ability of parent terpyridine ligands and their copper complexes to inhibit proteasome activity as an antineoplastic mechanism.

Hyaluronan-Cyclodextrin Conjugates as Doxorubicin Delivery Systems.

In the last years, nanoparticles based on cyclodextrins have been widely investigated for the delivery of anticancer drugs. In this work, we synthesized nanoparticles with a hyaluronic acid backbone functionalized with cyclodextrins under green conditions. We functionalized hyaluronic acid with two different molecular weights (about 11 kDa and 45 kDa) to compare their behavior as doxorubicin delivery systems. We found that the new hyaluronan-cyclodextrin conjugates increased the water solubility of doxorubicin. Moreover, we tested the antiproliferative activity of doxorubicin in the presence of the new cyclodextrin polymers in SK-N-SH and SK-N-SH-PMA (over-expressing CD44 receptor) cancer cells. We found that hyaluronan-cyclodextrin conjugates improved the uptake and antiproliferative activity of doxorubicin in the SK-N-SH-PMA compared to the SK-N-SH cell line at the ratio 8/1 doxorubicin/polymer. Notably, the system based on hyaluronan (45 kDa) was more effective as a drug carrier and significantly reduced the IC50 value of doxorubicin by about 56%. We also found that hyaluronic acid polymers determined an improved antiproliferative activity of doxorubicin (IC50 values are on average reduced by about 70% of free DOXO) in both cell lines at the ratio 16/1 doxorubicin/polymer.

Lubeluzole Repositioning as Chemosensitizing Agent on Multidrug-Resistant Human Ovarian A2780/DX3 Cancer Cells.

In a previous paper, we demonstrated the synergistic action of the anti-ischemic lubeluzole (Lube S) on the cytotoxic activity of doxorubicin (Dox) and paclitaxel in human ovarian cancer A2780 and lung cancer A549 cells. In the present paper, we extended in vitro the study to the multi-drug-resistant A2780/DX3 cell line to verify the hypothesis that the Dox and Lube S drug association may potentiate the antitumor activity of this anticancer compound also in the context of drug resistance. We also evaluated some possible mechanisms underlying this activity. We analyzed the antiproliferative activity in different cancer cell lines. Furthermore, apoptosis, Dox accumulation, MDR1 downregulation, ROS, and NO production in A2780/DX3 cells were also evaluated. Our results confirm that Lube S improves Dox antiproliferative and apoptotic activities through different mechanisms of action, all of which may contribute to the final antitumor effect. Moderate stereoselectivity was found, with Lube S significantly more effective than its enantiomer (Lube R) and the corresponding racemate (Lube S/R). Docking simulation studies on the ABCB1 Cryo-EM structure supported the hypothesis that Lube S forms a stable MDR1-Dox-Lube S complex, which hampers the protein transmembrane domain flipping and blocks the efflux of Dox from resistant A2780/DX3 cells. In conclusion, our in vitro studies reinforce our previous hypothesis for repositioning the anti-ischemic Lube S as a potentiating agent in anticancer chemotherapy.

Terpyridine functionalized cyclodextrin nanoparticles: metal coordination for tuning anticancer activity.

Multi-metal and multi-cavity systems based on the coordination properties of terpyridine functionalized cyclodextrin polymers were synthesized and characterized. Nanoparticles decorated with terpyridine derivatives via metal coordination showed high antiproliferative activity in tumor cells.

Cyclodextrin Polymers as Delivery Systems for Targeted Anti-Cancer Chemotherapy.

In the few last years, nanosystems have emerged as a potential therapeutic approach to improve the efficacy and selectivity of many drugs. Cyclodextrins (CyDs) and their nanoparticles have been widely investigated as drug delivery systems. The covalent functionalization of CyD polymer nanoparticles with targeting molecules can improve the therapeutic potential of this family of nanosystems. In this study, we investigated cross-linked γ- and ß-cyclodextrin polymers as carriers for doxorubicin (ox) and oxaliplatin (Oxa). We also functionalized γ-CyD polymer bearing COOH functionalities with arginine-glycine-aspartic or arginine moieties for targeting the integrin receptors of cancer cells. We tested the Dox and Oxa anti-proliferative activity in the presence of the precursor polymer with COOH functionalities and its derivatives in A549 (lung, carcinoma) and HepG2 (liver, carcinoma) cell lines. We found that CyD polymers can significantly improve the antiproliferative activity of Dox in HepG2 cell lines only, whereas the cytotoxic activity of Oxa resulted as enhanced in both cell lines. The peptide or amino acid functionalized CyD polymers, loaded with Dox, did not show any additional effect compared to the precursor polymer. Finally, studies of Dox uptake showed that the higher antiproliferative activity of complexes correlates with the higher accumulation of Dox inside the cells. The results show that CyD polymers could be used as carriers for repositioning classical anticancer drugs such as Dox or Oxa to increase their antitumor activity.

Blocking glutamate mGlu5 receptors with the negative allosteric modulator CTEP improves disease course in SOD1G93A mouse model of amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: The pathogenesis of amyotrophic lateral sclerosis (ALS) is not fully clarified, although excessive glutamate (Glu) transmission and the downstream cytotoxic cascades are major mechanisms for motor neuron death. Two metabotropic glutamate receptors (mGlu1 and mGlu5 ) are overexpressed in ALS and regulate cellular disease processes. Expression and function of mGlu5 receptors are altered at early symptomatic stages in the SOD1G93A mouse model of ALS and knockdown of mGlu5 receptors in SOD1G93A mice improved disease progression. EXPERIMENTAL APPROACH: We treated male and female SOD1G93A mice with 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), an orally available mGlu5 receptor negative allosteric modulator (NAM), using doses of 2 mg·kg-1 per 48 h or 4 mg·kg-1 per 24 h from Day 90, an early symptomatic disease stage. Disease progression was studied by behavioural and histological approaches. KEY RESULTS: CTEP dose-dependently ameliorated clinical features in SOD1G93A mice. The lower dose increased survival and improved motor skills in female mice, with barely positive effects in male mice. Higher doses significantly ameliorated disease symptoms and survival in both males and females, females being more responsive. CTEP also reduced motor neuron death, astrocyte and microglia activation, and abnormal glutamate release in the spinal cord, with equal effects in male and female mice. No differences were also observed in CTEP access to the brain. CONCLUSION AND IMPLICATIONS: Our results suggest that mGlu5 receptors are promising targets for the treatment of ALS and highlight mGlu5 receptor NAMs as effective pharmacological tools with translational potential.

Exploring Charged Polymeric Cyclodextrins for Biomedical Applications.

Over the years, cyclodextrin uses have been widely reviewed and their proprieties provide a very attractive approach in different biomedical applications. Cyclodextrins, due to their characteristics, are used to transport drugs and have also been studied as molecular chaperones with potential application in protein misfolding diseases. In this study, we designed cyclodextrin polymers containing different contents of ß- or γ-cyclodextrin, and a different number of guanidinium positive charges. This allowed exploration of the influence of the charge in delivering a drug and the effect in the protein anti-aggregant ability. The polymers inhibit Amiloid ß peptide aggregation; such an ability is modulated by both the type of CyD cavity and the number of charges. We also explored the effect of the new polymers as drug carriers. We tested the Doxorubicin toxicity in different cell lines, A2780, A549, MDA-MB-231 in the presence of the polymers. Data show that the polymers based on γ-cyclodextrin modified the cytotoxicity of doxorubicin in the A2780 cell line.

Lubeluzole: from anti-ischemic drug to preclinical antidiarrheal studies.

BACKGROUND: Lubeluzole, a neuroprotective anti-ischemic drug, was tested for its ability to act as both antibiotic chemosensitizing and antipropulsive agent for the treatment of infectious diarrhea. METHODS: In the present report, the effect of lubeluzole against antidiarrheal target was tested. The antimicrobial activity towards Gram-positive and Gram-negative bacteria was investigated together with its ability to affect ileum and colon contractility. RESULTS: Concerning the antimicrobial activity, lubeluzole showed synergistic effects when used in combination with minocycline against four common Gram-positive and Gram-negative bacteria (Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922), although relatively high doses of lubeluzole were required. In ex vivo experiments on sections of gut smooth muscles, lubeluzole reduced the intestinal contractility in a dose-dependent manner, with greater effects observed on colon than on ileum, and being more potent than reference compounds otilonium bromide and loperamide. CONCLUSION: All above results identify lubeluzole as a possible starting compound for the development of a novel class of antibacterial adjuvants endowed with spasmolytic activity.

Polypyridine ligands as potential metallo-ß-lactamase inhibitors.

Bacteria have developed multiple resistance mechanisms against the most used antibiotics. In particular, zinc-dependent metallo-ß-lactamase producing bacteria are a growing threat, and therapeutic options are limited. Zinc chelators have recently been investigated as metallo-ß-lactamase inhibitors, as they are often able to restore carbapenem susceptibility. We synthesized polypyridyl ligands, N,N'-bis(2-pyridylmethyl)-ethylenediamine, N,N,N'-tris(2-pyridylmethyl)-ethylenediamine, N,N'-bis(2-pyridylmethyl)-ethylenediamine-N-acetic acid (N,N,N'-tris(2-pyridylmethyl)-ethylenediamine-N'-acetic acid, which can form zinc(II) complexes. We tested their ability to restore the antibiotic activity of meropenem against three clinical strains isolated from blood and metallo-ß-lactamase producers (Klebsiella pneumoniae, Enterobacter cloacae, and Stenotrophomonas maltophilia). We functionalized N,N,N'-tris(2-pyridylmethyl)-ethylenediamine with D-alanyl-D-alanyl-D-alanine methyl ester with the aim to increase bacterial uptake. We observed synergistic activity of four polypyridyl ligands with meropenem against all tested isolates, while the combination N,N'-bis(2-pyridylmethyl)-ethylenediamine and meropenem was synergistic only against New Delhi and Verona integron-encoded metallo-ß-lactamase-producing bacteria. All synergistic interactions restored the antimicrobial activity of meropenem, providing a significant decrease of minimal inhibitory concentration value (by 8- to 128-fold). We also studied toxicity of the ligands in two normal peripheral blood lymphocytes.

Enhanced therapeutic index of liposomal doxorubicin Myocet locally delivered by fibrin gels in immunodeficient mice bearing human neuroblastoma.

Caelyx and Myocet are clinically used liposomal forms of doxorubicin (Dox). To explore ways to improve their therapeutic index, we have studied their activity in vitro and in vivo when locally delivered by fibrin gels (FBGs). In vivo local toxic and anti-tumour activities of loaded FBGs were assessed in two immunodeficient mouse orthotopic human neuroblastoma (NB) models after application in the visceral space above the adrenal gland, either still tumour-bearing or after tumour removal. In parallel, in vitro assays were used to mimic the in vivo overlaying of FBGs on the tumour surface. FBGs were prepared with different concentrations of fibrinogen (FG) and clotted in the presence of Ca2+ and thrombin. The in vitro assays showed that FBGs loaded with Myocet possess a cytotoxic activity against NB cell lines generally greater than those loaded with free Dox or Caelyx. In vivo FBGs loaded with Myocet showed lower general and local toxicities as compared to gels loaded with Caelyx or free Dox, and also to free Dox administered i.v. (all treatments with Dox at 2.5 mg/Kg). The anti-tumour activity, evaluated in the two mouse orthotopic NB models of adjuvant and neo-adjuvant therapy, resulted in a better performance of FBGs loaded with Myocet compared to the other local (FBGs loaded with Caelyx or free Dox) or systemic (free Dox) treatments (administered at 2.5 and 5 mg/Kg Dox). Specifically, the application of FBGs at 40 mg/mL in the adjuvant model caused 92 % tumour volume reduction, while by the neo-adjuvant application of FBGs at 22 mg/mL a re-growing tumour volume reduction of 89 % was obtained. Taken together, our in vitro and in vivo results indicate a significantly higher activity for the FBGs loaded with Myocet. In particular, the lower toicity coupled with the higher anti-tumour activity on both the local treatment modalities strongly suggest a better therapeutic index when Myocet is administered through FBGs. Therefore, FBGs loaded with Myocet may be considered as a possible new tool for the loco-regional treatment of NB or even other tumour histotypes treatable by loco-regional chemotherapy.

Antiproliferative and apoptotic activity of new indazole derivatives as potential anticancer agents.

To develop potent and selective anticancer agents, a series of novel polysubstituted indazoles was synthesized and evaluated for their in vitro antiproliferative and apoptotic activities against two selected human cancer cell lines (A2780 and A549). Several compounds showed an interesting antiproliferative activity, with IC50 values ranging from 0.64 to 17 µM against both cell lines. The most active indazoles were then tested in different pharmacological dilution conditions, adding five new cell lines (A2780, A549, IMR32, MDA-MB-231, and T47D) as targets, confirming their antiproliferative activity. Furthermore, selected compounds were able to trigger apoptosis to a significant extent and to cause, in part, a block of cells in the S phase of the cell cycle, with a concomitant decrease of cells in the G2/M and/or G0/G1 phases and the generation of hypodiploid peaks. However, molecule 7d caused a great increase of cells in G2/M and the appearance of polyploid cells. Altogether, our results suggest a good pharmacological activity for our selected polysubstituted indazoles, which are suggestive of a preferential mechanism of action as cell cycle-specific antimetabolites or as an inhibitor of enzyme activities involved in DNA synthesis, except for 7d, which, on the contrary, seems to have a mechanism involving the microtubule system.

Discovery of New Antiproliferative Imidazopyrazole Acylhydrazones Able To Interact with Microtubule Systems.

Even though immunotherapy has radically changed the search for anticancer therapies, there are still many different pathways that are open to intervention with traditional small molecules. To expand our investigation in the anticancer field, we report here a new series of compounds in which our previous pyrazole and imidazopyrazole scaffolds are linked to a differently decorated phenyl ring through an acylhydrazone linker. Preliminary tests on the library were performed at the National Cancer Institute (USA) against the full NCI 60 cell panel. The best compounds among the imidazopyrazole series were then tested by immunofluorescence staining for their inhibition of cell proliferation, apoptosis induction, and their effect on the cell cycle and on microtubules. Two compounds, in particular 4-benzyloxy-3-methoxybenzyliden imidazopyrazole-7-carbohydrazide showed good growth inhibition, with IC50 values in the low-micromolar range, and induced apoptosis. Both compounds altered the cell-cycle phases with the appearance of polyploid cells. Immunofluorescence analysis evidenced microtubules alterations; tubulin polymerization assays and docking studies suggested the tubulin system to be the possible, although not exclusive, target of the new acylhydrazone series reported here.

3-Aryl-4-nitrobenzothiochromans S,S-dioxide: From Calcium-Channel Modulators Properties to Multidrug-Resistance Reverting Activity.

Our research groups have been involved for many years in studies aimed at identifying new active organic compounds endowed with pharmacological properties. In this work, we focused our attention on the evaluation of cardiovascular and molecular drug resistance (MDR) reverting activities of some nitrosubstituted sulphur-containing heterocycles. Firstly, we have examined the effects of 4-nitro-3-(4-methylphenyl)-3,6-dihydro-2H-thiopyran S,S-dioxide 5, and have observed no activity. Then we have extended our investigation to the 3-aryl-4-nitrobenzothiochromans S,S-dioxide 6 and 7, and have observed an interesting biological profile. Cardiovascular activities were assessed for all compounds using ex vivo studies, while the MDR reverting effect was evaluated only for selected compounds using tumor cell lines. All compounds were shown to affect cardiovascular parameters. Compound 7i exerted the most effect on negative inotropic activity, while 6d and 6f could be interesting molecules for the development of more active ABCB1 inhibitors. Both 6 and 7 represent structures of large possible biological interest, providing a scaffold for the identification of new ABCB1 inhibitors.

Fibrin gels entrapment of a doxorubicin-containing targeted polycyclodextrin: Evaluation of in vivo antitumor activity in orthotopic models of human neuroblastoma.

In vivo local antitumor activity of fibrin gels (FBGs) loaded with the poly-cyclodextrin oCD-NH2/Dox, compared to free Dox, was evaluated in two mouse orthotopic neuroblastoma (NB) models, after positioning of the releasing devices in the visceral space. FBGs were prepared at the fibrinogen (FG) concentrations of 22 and 40 mg/ml clotted in the presence of 0.81 mM/mg FG Ca2+ and 1.32 U/mg FG thrombin. Our results indicate that FBGs loaded with oCD-NH2/Dox and applied as neoadjuvant loco-regional treatment, show an antitumor activity significantly greater than that displayed by the same FBGs loaded with identical dose of Dox or after free Dox administered intra venous (iv). In particular, FBGs prepared at 40 mg/ml showed a slightly lower antitumor activity, although after their positioning we observed a significant initial reduction of tumor burden lasting for several days after gel implantation. FBGs at 22 mg/ml loaded with oCD-NH2/Dox and applied after tumor removal (adjuvant treatment model) showed a significantly better antitumor activity than the iv administration of free Dox, with 90% tumor regrowth reduction compared to untreated controls. In all cases the weight loss post-treatment was limited after gel application, although in the adjuvant treatment the loss of body weight lasted longer than in the other treatment modality. In accordance with our recent published data on the low local toxic effects of FBGs, the present findings also underline an increase of the therapeutic index of Dox when locally administered through FBGs loaded with the oCD-NH2/Dox complex.

Fibrin Gels Entrapment of a Poly-Cyclodextrin Nanocarrier as a Doxorubicin Delivery System in an Orthotopic Model of Neuroblastoma: Evaluation of In Vitro Activity and In Vivo Toxicity.

PURPOSE: Fibrin gels (FBGs) are potential delivery vehicles for many drugs, and can be easily prepared from purified components. We previously demonstrated their applicability for the release of different doxorubicin (Dox) nanoparticles used clinically or in an experimental stage, such as its inclusion complex with the amino ß-cyclodextrin polymer (oCD-NH2/Dox). Here we extend these studies by in vitro and in vivo evaluations. METHODS: An in vitro cytotoxicity model consisting of an overlay of a neuroblastoma (NB) cell-containing agar layer above a drug-loaded FBG layer was used. Local toxicity in vivo (histology and blood analysis) was studied in a mouse orthotopic NB model (SHSY5YLuc+ cells implanted into the left adrenal gland). RESULTS: In vitro data show that FBGs loaded with oCD-NH2/Dox have a slightly lower cytotoxicity against NB cell lines than those loaded with Dox. Fibrinogen (FG), and Ca2+ concentrations may modify this activity. In vivo data support a lower general and local toxicity for FBGs loaded with oCD-NH2/Dox than those loaded with Dox. CONCLUSION: Our results suggest a possible increase of the therapeutic index of Dox when locally administered through FBGs loaded with oCD-NH2/Dox, opening the possibility of using these releasing systems for the treatment of neuroblastoma.

Cyclodextrin polymers decorated with RGD peptide as delivery systems for targeted anti-cancer chemotherapy.

Polymeric cyclodextrin-based nanoparticles are currently undergoing clinical trials as nanotherapeutics. Using a non-covalent approach, we decorated two cross-linked cyclodextrin polymers of different molecular weights with an RGD peptide derivative to construct a novel carrier for the targeted delivery of doxorubicin. RGD is the binding sequence for the integrin receptor family that is highly expressed in tumour tissues. The assembled host-guest systems were investigated using NMR and DLS techniques. We found that, in comparison with free doxorubicin or the binary complex doxorubicin/cyclodextrin polymer, the RGD units decorating the cyclodextrin-based nanosystems improved the selectivity and cytotoxicity of the complexed doxorubicin towards cultured human tumour cell lines. Our results suggest that the nanocarriers under study may contribute to the development of new platforms for cancer therapy.

Characterization of drug release from fibrin gels loaded with different pharmaceutical and experimental doxorubicin formulations.

BACKGROUND: Local delivery of anticancer drugs represents a desirable type of treatment. Nevertheless, characteristics such as availability, biocompatibility, ease of operation, and efficacy sometimes represent difficult to overcome hurdles. Fibrin gels (FBGs) may be attractive biomaterials for local treatment when loaded with different chemotherapeutics or with polymer-anticancer-drug conjugates and nanoparticles. These components, linked together, might represent candidates to counteract local recurrences or reduce the volume of inoperable tumors. In the present study we analyzed the features of in vitro release of different formulations of doxorubicin (DOXO) from FBGs, and in vivo FBGs degradation. METHODS: In vitro DOXO release from FBGs was studied as a function of thrombin and Ca2+ ion concentrations. DOXO was loaded in FBGs either alone or pre-incorporated in nanoparticles characterized by different physical features. The FBGs in vivo degradation was analyzed after sc or ip positioning. RESULTS: Our results suggest that each of the factors involved in the FBGs preparation may have different effects on drug release. In particular, the fibrinogen (FG) concentration and, above all, the DOXO formulation were found to have the greatest impact. Not surprisingly, we have also found a longer permanence in vivo of FBGs prepared at the highest thrombin, Ca2+ ion, and FG concentrations. CONCLUSIONS: The aim of this work was to study the effect of several conditions for preparing drug delivery systems based on FBGs loaded with different clinical or experimental formulations of DOXO. Our data identify some of these modalities that will be tested in vivo to evaluate their antitumor activity.