Synthesis and Evaluation of new prodrug systems of the natural polyphenol Curcumin

The thesis reports the synthesis and characterization of new carbamoylic prodrugs of the natural bioactive polyphenol Curcumin, found in many foodstuff. Curcumin is a promising drug candidate because of its credited beneficial health effects and wide spectrum of action. However, the low bioavailability due to its poor solubility in aqueous media (i.e. physiological fluids) and to its very fast hepatic metabolism, greatly limits the use of Curcumin in the pharmaceutical field. The “prodrug approach” represents a valid solution to overcome these issues. This is based on the conjugation of the active compound with a specific promoiety through a bioreversible linkage. This modification is expected to improve the pharmacokinetic profile of the compound (prevent fast metabolism and enhance bioavailability). The choice of the bioreversible linker is crucial for the pharmacokinetic performance of the prodrug. I chose and used a urethane linkage, which has been recently demonstrated by our group to be the best option to protect and enhance the bioavailability of natural polyphenols in vivo. Curcumin was conjugated with two different types of promoieties: natural amino acids and poly-2-alkyl-oxazolines, a class of hydrophilic synthetic polymers. In particular, leucine, isoleucine and valine were selected as promoieties for the conjugation with Curcumin, since these amino acids show enhanced intestinal absorption due to active transport mechanism. The conjugation of these amino acids with Curcumin could thus improve the absorption of Curcumin following oral administration of the prodrug. Curcumin has two phenolic hydroxyl groups. I functionalized Curcumin on one and both hydroxy groups by conjugation with amino acids through a urethane linkage, obtaining six new prodrugs (mono- and di-substituted, respectively). The stability of the new prodrugs and of Curcumin itself in aqueous media was studied using different experimental approaches: UPLC-UV, HPLC/MS, UV-Vis spectroscopy and 1H-NMR. The study of these processes and systems was severely hindered by the solubility of the new Curcumin prodrugs in aqueous media which turned out to be rather poor, either under acidic or near neutral pH conditions, as are found in the stomach and intestine. The prodrugs proved to be more stable in acidic solutions as expected based on the known reactivity of the N-monosubstituted carbamoylic linkage. In solutions at pH 6.8 (as found in the intestine), some degradation was observed. The stability of the new derivatives against hydrolysis was also studied by 1H-NMR spectroscopy in D2O-DMSO-d6 solutions with different D2O contents. No degradation was observed in these aqueous media over three hours. Preliminary pharmacokinetic experiments were carried out and proved that the di-substituted leucine carbamoyl derivative of Curcumin is capable of improving the bioavailability of the parent phenol in mice model. Finally, the affinity of these prodrugs for plasma proteins was assed, using fluorescence binding assays with human serum albumin (HSA), which is ubiquitous in the blood stream. All of the new derivatives, except for the mono-leucine prodrug, have higher affinity for HSA than Curcumin itself. From these combined data a good distribution in the blood stream and high stability against hepatic enzymes can be expected for the new derivatives developed with this thesis, in particular for the mono-functionalized compounds, which make these compounds potential candidates as Curcumin prodrugs. The last chapter of this thesis is focused on the synthesis of new drug-delivery systems obtained from the self-assembly of amphiphilic polymeric prodrugs of Curcumin. Also in this case a carbamoylic bond was chosen as the linkage between Curcumin and the polymer chain. Poly-2-methyl-2-oxazolines (PMOXAs) were chosen as promoieties for conjugation with Curcumin because of their similarity to the most commonly used poly-ethylene glycols (PEGs), showing the same stealth effects, together with a reduced immune response, and a higher hydrophilicity. In particular, five poly-2-methyl-2-oxazolines of different chain length were conjugated to one hydroxy group of Curcumin. Because of the high hydrophilic character of the PMOXAs, these five new prodrugs can be classified as amphiphiles. Indeed they possess the ability to self-assemble in micelle-like structures when put in aqueous solution, providing good-to-excellent solubility of Curcumin in physiological-like media. The correlation between the chain length and the self-assembly ability of these prodrugs was investigated. All derivatives have a critical micellar concentration (CMC) in the 10-6-10-4 M range. Curcumin-PMOXA30 showed the highest ability to self assemble into micelles at concentrations as low as 10-6 M. On the contrary, conjugates with PMOXA50 and PMOXA100 resulted in a higher CMC value, probably due to the unfavourable hydrophilic/hydrophobic ratio, typical of longer polymeric chain, which prevents the self-assembly process. On the other hand the derivative Curcumin-PMOXA10, showed the tendency to aggregate and precipitate in solution, probably due to a too pronounced hydrophobic character. The size and the stability in water of all micelles were tested using dynamic light scattering (DLS) and transmission electron microscopy (TEM). An inverse correlation between chain length and size was found. In fact, conjugates with PMOXA20 and PMOXA30 form micelles with a diameter around 100-130 nm, whereas analogues with PMOXA50 and PMOXA100 with diameters of about 50-30 nm. For all conjugates, except Curcumin-PMOXA10, a good stability in aqueous environments was observed. Furthermore, I tested the release of Curcumin from each type of micelles by means of UV-Vis analysis. The experiments were carried out in phosphate buffered saline (PBS) at pH 7.4 and at 37°C in order to mimic the blood pH value. The conjugate Curcumin-PMOXA30 showed the best kinetic profile with around 90% of released Curcumin during five hours. The other conjugates showed instead a release of Curcumin around 10-30% during the same time.

Free-living marine nematodes from San Antonio Bay (Río Negro, Argentina)

The dataset of free-living marine nematodes of San Antonio Bay is based on sediment samples collected in February 2009 during doctoral theses funded by CONICET grants. A total of 36 samples has been taken at three locations in the San Antonio Bay, Santa Cruz Province, Argentina on the coastal littoral at three tidal levels. This presents a unique and important collection for benthic biodiversity assessment of Patagonian nematodes as this area remains one of the least known regions. In total 7,743 specimens of free-living marine nematodes belonging to two classes, eight orders, 37 families, 94 genera and 104 species were collected.Fil: Villares, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Lo Russo, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Diversidad y Evolución Austral; ArgentinaFil: Pastor, Catalina Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Diversidad y Evolución Austral; ArgentinaFil: Milano, Viviana. Universidad Nacional de la Patagonia; ArgentinaFil: Miyashiro, Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Mazzanti, Renato. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; Argentin

Prognostic and predictive factors in colorectal cancer: Kirsten Ras in CRC (RASCAL) and TP53CRC collaborative studies.

Mutations in the Ki-ras and TP53 genes are the most frequently observed genetic alterations in colorectal cancer (CRC). Ki-ras mutations are mostly found in codons 12 and 13, and less in codon 61. The majority of the TP53 mutations occur in the core domain which contains the sequence-specific DNA binding activity of the protein, and they results in loss of DNA binding. Few centres have sufficient patients to collect detailed information in the large numbers required to determine the impact of individual ki-ras and TP53 genotypes on outcome. Moreover, it has been reported that specific genetic alterations, and not any mutation, might play a different biological role in cancer progression. For these principal reasons, two collaborative studies have been conducted (the RASCAL and the TP53-CRC Collaborative Studies) with the aim of investigating the prognostic role of any, and specific, Ki-ras and TP53 mutations in CRC progression. The results obtained from the RASCAL studies suggest that Ki-ras mutations might have an effect on the survival rate of CRC patients, and that the specific codon 12 glycine/valine mutation might play a role in the progression of this neoplasia. The results of the TP53-CRC International Collaborative Study demonstrate the importance of primary tumor site when analyzing the prognostic value of TP53 mutations in CRC. In addition, different types of TP53 mutation might play a pivotal role in determining the biological behavior of CRC from different sites and hence the prognosis of patients. This meta-analysis produced evidence for interesting tumor site differences in the predictive value of TP53 mutation for survival benefit from 5FU chemotherapy

Apoptosis: a relevant tool for anticancer therapy.

Apoptosis is a form of cell death that permits the removal of damaged, senescent or unwanted cells in multicellular organisms, without damage to the cellular microenvironment. Defective apoptosis represents a major causative factor in the development and progression of cancer. The majority of chemotherapeutic agents, as well as radiation, utilize the apoptotic pathway to induce cancer cell death. Resistance to standard chemotherapeutic strategies also seems to be due to alterations in the apoptotic pathway of cancer cells. Recent knowledge on apoptosis has provided the basis for novel targeted therapies that exploit apoptosis to treat cancer. These new target include those acting in the extrinsic/intrinsic pathway, proteins that control the apoptosis machinery such as the p53 and proteosome pathway. Most of these forms of therapy are still in preclinical development because of their low specifity and susceptibility to drug resistance, but several of them have shown promising results. In particular, this review specifically aims at providing an update of certain molecular players that are already in use in order to target apoptosis (such as bortezomib) or which are still being clinically evaluated (such ONYX-015, survivin and exisulind/aptosyn) or which, following preclinical studies, might have the necessary requirements for becoming part of the anticancer drug programs (such as TRAIL/Apo2L, apoptin/VP3)

Male breast cancer.

Male breast cancer (MaleBC) is a rare disease, accounting for <1% of all male tumors. During the last few years, there has been an increase in the incidence of this disease, along with the increase in female breast cancer (FBC). Little is known about the etiology of MaleBC: hormonal, environmental and genetic factors have been reported to be involved in its pathogenesis. Major risk factors include clinical disorders carrying hormonal imbalances, radiation exposure and, in particular, a positive family history (FH) for BC, the latter suggestive of genetic susceptibility. Rare mutations in high-penetrance genes (BRCA1 and BRCA2) confer a high risk of BC development; low-penetrance gene mutations (i.e. CHEK-2) are more common but involve a lower risk increase. About 90% of all male breast tumors have proved to be invasive ductal carcinomas, expressing high levels of hormone receptors with evident therapeutic returns. The most common clinical sign of BC onset in men is a painless palpable retroareolar lump, which should be evaluated by means of mammography, ultrasonography and core biopsy or fine needle aspiration (FNA). To date, there are no published data from prospective randomized trials supporting a specific therapeutic approach in MaleBC. Tumor size together with the number of axillary nodes involved are the main prognostic factors and should guide the treatment choice. Locoregional approaches include surgery and radiotherapy (RT), depending upon the initial clinical presentation. When systemic treatment (adjuvant, neoadjuvant and metastatic) is delivered, the choice between hormonal and or chemotherapy (CT) should depend upon the clinical and biological features, according to the FBC management guidelines. However great caution is required because of high rates of age-related comorbidities

Can KRAS and BRAF mutations limit the benefit of liver resection in metastatic colorectal cancer patients? A systematic review and meta-analysis

Clinical trials investigated the potential role of both KRAS and BRAF mutations, as prognostic biomarkers, in colorectal cancer (CRC) patients who underwent surgical treatment of CRC-related liver metastases (CLM), showing conflicting results. This meta-analysis aims to review all the studies reporting survival outcomes (recurrence free survival (RFS), and/or overall survival (OS)) of patients undergoing resection of CLM, stratified according to KRAS and/or BRAF mutation status.Background: Clinical trials investigated the potential role of both KRAS and BRAF mutations, as prognostic biomarkers, in colorectal cancer (CRC) patients who underwent surgical treatment of CRC-related liver metastases (CLM), showing conflicting results. This meta-analysis aims to review all the studies reporting survival outcomes (recurrence free survival (RFS), and/or overall survival (OS)) of patients undergoing resection of CLM, stratified according to KRAS and/or BRAF mutation status. Materials and methods: Data from all published studies reporting survival outcomes (RFS and/or OS) of CRC patients who received resection of CLM, stratified by KRAS and/or BRAF mutation status were collected, according to the PRISMA guidelines. Pooled HRs were calculated for both the OS and/or RFS. Results: Seven eligible trials (1403 patients) were included. Pooled analysis showed that KRAS mutations predicted a significantly worse both RFS (HR: 1.65; 95% CI: 1.23-2.21) and OS (HR: 1.86; 95% CI: 1.51-2.30) in patients who underwent surgical resection of CLM. BRAF mutations were also associated with a significantly worse OS (HR: 3.90; 95% CI: 1.96-7.73) in this subgroup of patients. Conclusions: This meta-analysis suggests both KRAS and BRAF mutations as poor, prognostic biomarkers, associated with worse survival outcomes, in patients undergoing hepatic resection of CLM

Hereditary ovarian cancer

Apoptosis is a form of cell death that permits the removal of damaged, senescent or unwanted cells in multicellular organisms, without damage to the cellular microenvironment. Defective apoptosis represents a major causative factor in the development and progression of cancer. The majority of chemotherapeutic agents, as well as radiation, utilize the apoptotic pathway to induce cancer cell death. Resistance to standard chemotherapeutic strategies also seems to be due to alterations in the apoptotic pathway of cancer cells. Recent knowledge on apoptosis has provided the basis for novel targeted therapies that exploit apoptosis to treat cancer. These new target include those acting in the extrinsic/intrinsic pathway, proteins that control the apoptosis machinery such as the p53 and proteosome pathway. Most of these forms of therapy are still in preclinical development because of their low specifity and susceptibility to drug resistance, but several of them have shown promising results. In particular, this review specifically aims at providing an update of certain molecular players that are already in use in order to target apoptosis (such as bortezomib) or which are still being clinically evaluated (such ONYX-015, survivin and exisulind/aptosyn) or which, following preclinical studies, might have the necessary requirements for becoming part of the anticancer drug programs (such as TRAIL/ Apo2L, apoptin/VP3). Key words: apoptosis, TRAIL/Apo2L, apoptin/VP3, ONYX015, Bortezomib, exisulind, survivi

Breast cancer genome-wide association studies: there is strength in numbers

Breast cancer (BC) is a heterogeneous disease that exhibits familial aggregation. Family linkage studies have identified high-penetrance genes, BRCA1, BRCA2, PTEN and TP53, that are responsible for inherited BC syndromes. Moreover, a combination of family-based and population-based approaches indicated that genes involved in DNA repair, such as CHEK2, ATM, BRIP and PALB2, are associated with moderate risk. Therefore, all of these known genes account for only 25% of the familial aggregation cases. Recently, genome wide association studies (GWAS) in BC revealed single nucleotide polymorphisms (SNPs) in five novel genes associated to susceptibility: TNRC9, FGFR2, MAP3K1, H19 and lymphocyte-specific protein 1 (LSP1). The most strongly associated SNP was in intron 2 of the FGFR2 gene that is amplified and overexpressed in 5-10% of BC. rs3803662 of TNRC9 gene has been shown to be the SNP with the strongest association with BC, in particular, this polymorphism seems to be correlated with bone metastases and estrogen receptor positivity. Relevant data indicate that SNP rs889312 in MAP3K1 is correlated with BC susceptibility only in BRCA2 mutation carriers, but is not associated with an increased risk in BRCA1 carriers. Finally, different SNPs in LSP1 and H19 and in minor genes probably were associated with BC risk. New susceptibility allelic variants associated with BC risk were recently discovered including potential causative genes involved in regulation of cell cycle, apoptosis, metabolism and mitochondrial functions. In conclusion, the identification of disease susceptibility loci may lead to a better understanding of the biological mechanism for BC to improve prevention, early detection and treatment

Looking for the best immune-checkpoint inhibitor in pre-treated NSCLC patients: An indirect comparison between nivolumab, pembrolizumab and atezolizumab

Immune-checkpoint inhibitors represent the new standard of care in patients with advanced NSCLC who progressed after first-line treatment. This work aim to assess any difference in both efficacy and safety profiles among Nivolumab, Pembrolizumab and Atezolizumab in pre-treated NSCLC patients. Randomized clinical trials comparing immune-checkpoint inhibitor versus docetaxel in pre-treated patients with advanced NSCLC were included and direct comparison meta-analysis of selected trials have been performed. Subsequently the summary estimates of Nivolumab, Pembrolizumab and Atezolizumab emerging from the direct meta-analysis were selected to provide the pooled estimates of hazard ratio (HR) and relative risk (RR) for the indirect comparisons among these agents. A total of 5 studies met the selection criteria and were included in the meta-analysis. Indirect comparisons for efficacy outcomes showed the RR for ORR nivolumab versus atezolizumab 1.66 (95% CI 1.07â2.58), pembrolizumab versus atezolizumab 1.94 (95% CI 1.30â2.90). No significant differences in both PFS and OS have been observed. Indirect comparisons for safety showed the RR for G3-5 AEs nivolumab versus pembrolizumab 0.41 (95% CI 0.29â0.60), nivolumab versus atezolizumab 0.50 (95% CI 0.35â0.72). No significant differences in both pneumonitis and discontinuation rate have been observed. The results of this work revealed that nivolumab and pembrolizumab are associated with a significant increase of ORR as compared to atezolizumab and nivolumab is associated with a significant lower incidence of G3-5 AEs as compared to the other drugs. These evidences could support the oncologists to select the best drug for each patient

Free-living marine nematodes from San Julián Bay (Santa Cruz, Argentina)

The free-living marine nematodes of San Julián Bay dataset is based on sediment samples collected in January 2009 during the project PICT AGENCIA-FONCYT 2/33345-2005. A total of 36 samples have been taken at three locations in the San Julián Bay, Santa Cruz Province, Argentina on the coastal littoral at three tidal levels. This presents a unique and important collection for the nematode benthic biodiversity assessment as this area remains one of the least known regions in Patagonia. In total 10,030 specimens of free-living marine nematodes belonging to 2 classes, 9 orders, 35 families, 78 genera and 125 species were collected. The San Julián city site presented a very high species richness.Fil: Pastor, Catalina Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Lo Russo, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Villares, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Milano, Viviana. Universidad Nacional de la Patagonia; ArgentinaFil: Miyashiro, Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Mazzanti, Renato. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; Argentin