Small molecules exploiting emerging therapeutic opportunities for breast cancer treatment

Among the different types of breast cancer (BC), the estrogen receptor positive (ER+) subtype, which requires estrogens for its growth and proliferation, is the most common, while triple negative BC, characterized by the absence of ER, progesterone receptor and human epidermal growth factor receptor 2, often leads to poor prognosis. First-line therapies for the treatment of ER+ BC act either by suppressing estrogen production, through the inhibition of aromatase (AR) enzyme, or by blocking estrogen prooncogenic activity, via the modulation/degradation of ERs. The serious side effects and the intrinsic or acquired resistance phenomena that arise with prolonged use of these drugs limit their therapeutic application, stimulating the search for new strategies to face this disease. In this context, the development of dual acting aromatase inhibitors, able to target both the orthosteric and the recently identified allosteric pockets of AR could be an opportunity to fight ER+ BC. Another promising strategy could be the development of multitarget compounds, targeting both AR and ERs. In this scenario, here we designed and synthesized two series of new xanthones or more flexible benzophenones as potential dual acting aromatase inhibitors. Moreover, inspired from tamoxifen metabolites and a literature compound endowed with activity on both AR and ER, different structurally related series of potential multitarget compounds were developed. The biological results showed that some of the new molecules were promising candidates for further development. It was recently observed that the lately discovered histamine H4 receptor is expressed in human breast tissue, displaying a key role in biological processes mediated by histamine such as cell proliferation, senescence, and apoptosis in malignant cells, representing a potential target in triple negative BC. Thus, a broad series of methyl quinazoline sulfonamides, carrying different functional groups on the sulfonamide moiety, were designed and synthesized as potential H4 receptor ligands.

Molecular target therapy and immunotherapy of rare lung tumors

Lung cancer is an heterogeneous disease, with 1-2% of rare histology. New molecular profiling technologies, such as next generation sequencing (NGS), haverevolutionized the assessment of molecular alteration in clinical practice. We analyzed a cohort of 1408 NSCLC-A patients treated at the Sant'Orsola- Malpighi University Hospital from 2019 to 2021. This analysis was performed using the oncomine focus thermo fischer panel. Of them, 410 (29%) had rare alteration (RET 3%, NTRK 0,2%,FGFR1 2%, MET exon14 skipping 3%, BRAF V600 4%, ALK fusion EGFR exon 20 2%) and 36 (2%)had a uncommon mutation. We enrolled 7 RET- rearranged patients in CRETA and J2G-MC-JZJC clinical trials assessing respectively unselective and selective RET-inhibitors , another 7 patients tested positive for the BRAF V6006 mutation and have been enrolled in the Array clinical trial assessing a novel combination of anti-BRAF and anti-mek agents . Other molecular alterations found are KRAS (Gly12Cys), FGFR1-4 mutation, MET skipping ex14 mutations, respectively eligible for other ongoing open studies such as Amgen 20190009 comparing efficacy of sotorasib vs docetaxel, Fight-207 assessing activity of pemigatinib and CINC280J12201 assessing activity of the novel met inhibitor capmatinib. In 2018 we joined the CHANCE clinical trial,a multicenter study evaluating the efficacy and safety of atezolizumab in patients withrare lung cancer histologies where and 14 patients have been so far enrolled in the Bologna site. Our studies underline the need of tailored approach to NSCLC patients and our results showed that precision medicine is feasible and is an effective approach to cancer treatment.

Development of new chemical entities to target neuroinflammatory pathways.

Neuroinflammatory pathways are main culprits of neurodegenerative diseases' onset and progression, including Alzheimer’s disease (AD). On this basis, several anti-inflammatory drugs were repurposed in clinical trials. However, they have failed, probably because neuroinflammation is a complex network, still not fully understood. From these evidences, this thesis focused on the design and synthesis of new chemical entities as potential neuroinflammatory drugs or chemical probes. Projects 1 and 2 aimed to multi-target-directed ligand (MTDL) development to target neuroinflammation in AD. Polypharmacology by MTDLs is considered one of the most promising strategies to face the multifactorial nature of neurodegenerative diseases. Particularly, Project 1 took inspiration from a cromolyn-ibuprofen drug combination polypharmacological approach, which was recently investigated in AD clinical trials. Based on that, two cromolyn-(S)-ibuprofen codrug series were designed and synthesized. Parent drugs were combined via linking or fusing strategies in 1:2 or 1:1 ratio, by means of hydrolyzable bonds. Project 2 started from a still ongoing AD clinical trial on investigational drug neflamapimod. It is a selective inhibitor of p38α-MAPK, a kinase strictly involved in neuroinflammatory pathways. On the other side, rasagiline, an anti-Parkinson drug, was also repurposed as AD treatment. Indeed, rasagiline’s propargylamine fragment demonstrated to be responsible not only for the MAO-B selective inhibition, but also for the neuroprotective activity. Thus, to synergistically combine these two effects into single-molecules, a small set of neflamapimod-rasagiline hybrids was developed. In the end BMX, a poorly investigated kinase, which seems to be involved in pro-inflammatory mediator production, was explored for the development of new chemical probes. High-quality chemical probes are a powerful tool in target validation and starting points for the development of new drug candidates. Thus, Project 3 focused on the design and synthesis of two series of optimized BMX covalent inhibitors as selective chemical probes.

Research of predictive biomarkers to anti-CD22 antibody-drug conjugate treatment in B-cell acute lymphoblastic leukemia

Background: The treatment of B-cell acute lymphoblastic leukemia (B-ALL) has been enriched by novel agents targeting surface markers CD19 and CD22. Inotuzumab ozogamicin (INO) is a CD22-calicheamicin conjugated monoclonal antibody approved in the setting of relapse/refractory (R/R) B-ALL able to induce a high rate of deep responses, not durable over time. Aims: This study aims to identify predictive biomarkers to INO treatment in B- ALL by flow cytometric analysis of CD22 expression and gene expression profile. Materials and methods: Firstly, the impact on patient outcome in 30 R/R B-ALL patients of baseline CD22 expression in terms of CD22 blast percentage and CD22 fluorescent intensity (CD22-FI) was explored. Secondly, baseline gene expression profile of 18 R/R B-ALL patient samples was analyzed. For statistical analysis of differentially expressed genes (DEGs) patients were divided in non-responders (NR), defined as either INO-refractory or with duration of response (DoR) < 3 months, and responders (R). Gene expression results were analyzed with Ingenuity pathway analysis (IPA). Results: In our patient set higher CD22-FI, defined as higher quartiles (Q2-Q4), correlated with better patient outcome in terms of CR rate, OS and DoR, compared to lower CD22-FI (Q1). CD22 blast percentage was less able to discriminate patients’ outcome, although a trend for better outcome in patients with CD22 ≥ 90% could be appreciated. Concerning gene expression profile, 32 genes with corrected p value <0.05 and absolute FC ≥2 were differentially expressed in NR as compared to R. IPA upstream regulator and regulator effect analysis individuated the inhibition of tumor suppressor HIPK2 as causal upstream condition of the downregulation of 6 DEGs. Conclusions: CD22-FI integrates CD22-percentage on leukemic blasts for a more comprehensive target pre-treatment evaluation. Moreover, a unique pattern of gene expression signature based on HIPK2 downregulation was identified, providing important insights in mechanisms of resistance to INO.

Appropriate technologies and natural systems for wastewater treatment in low- and middle-income countries

Wastewater management is an environmental and social burden that primarily affects populations in Low- and Middle-Income Countries and the global environment. Wastewater collection, treatment, and reuse have become urgent, especially considering that 80% of the world's wastewater is untreated or improperly treated and discharged directly into water bodies. In recent years, the role of wastewater treatment plants in a sustainable water cycle has become even more critical, as they are the final destination of the collected wastewater. Indeed, the management of wastewater treatment plants should play an essential role in achieving SDG target 6.3 of the United Nations 2030 Agenda for SD. In this context, water reuse, especially wastewater reuse, plays a key role. This research focuses on investigating the valorization of wastewater resources applying Appropriate Technologies and Natural Systems for wastewater treatment in two different Low- and Middle-Income Countries, the Palestinian Territories and Sub-Saharan Africa. The research objectives are: (1) Determine the characteristics and quality of wastewater in the two case studies analysed. (2) Identify Appropriate Technology to be used in the Palestinian Territories to treat wastewater for reuse in agriculture. (3) Assess the environmental, economic, and social impacts of this project. (4) Assess the feasibility of using natural wetlands for household wastewater treatment in Sub-Saharan region. The first study, conducted in Rafah, Gaza Strip, showed that implementing existing primary treatment plant with a natural secondary treatment plant properly optimized the wastewater quality for reuse in agriculture and was suitable for the study area. The second case study was conducted in Cape Coast, Ghana. It shows that the natural wetland studied is currently overly polluted and threatened by various anthropogenic factors that cannot remove pollutants from the incoming domestic wastewater. Therefore, some recommendations were made in order to improve the efficiency of this natural wetland.

Harnessing the cellular and molecular complexity of high-risk neuroblastoma to investigate novel potential treatment approaches

Neuroblastoma (NB) is the most common type of tumor in infants and the third most common cancer in children. Current clinical practices employ a variety of strategies for NB treatment, ranging from standard chemotherapy to immunotherapy. Due to a lack of knowledge about the molecular mechanisms underlying the disease's onset, aggressive phenotype, and therapeutic resistance, these approaches are ineffective in the majority of instances. MYCN amplification is one of the most well-known genetic alterations associated with high risk in NB. The following work is divided into three sections and aims to provide new insights into the biology of NB and hypothetical new treatment strategies. First, we identified RUNX1T1 as a key gene involved in MYCN-driven NB onset in a transgenic mouse model. Our results suggested that that RUNX1T1 may recruit the Co-REST complex on target genes that regulate the differentiation of NB cells and that the interaction with RCOR3 is essential. Second, we provided insights into the role of MYCN in dysregulating the CDK/RB/E2F pathway controlling the G1/S transition of the cell cycle. We found that RB is dispensable in regulating MYCN amplified NB's cell cycle, providing the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression. Third, we generated an M13 bacteriophage platform to target GD2-expressing cells in NB. Here, we generated a recombinant M13 phage capable of binding GD2-expressing cells selectively (M13GD2). Our results showed that M13GD2 chemically conjugated with the photosensitizer ECB04 preserves the retargeting capability, inducing cell death even at picomolar concentrations upon light irradiation. These results provided proof of concept for M13 phage employment in targeted photodynamic therapy for NB, an exciting strategy to overcome resistance to classical immunotherapy.

Preclinical evaluation of a MYCN-specific inhibitor for the treatment of small cell lung cancer

Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, characterized by rapid growth, early metastasis and acquired drug resistance. SCLC is usually sensitive to initial treatment, however, most patients relapse within few months; thus more effective therapies are urgently needed. Key genetic alterations very frequently observed in SCLC include loss of TP53 and RB1 and mutations in the MYC family genes (MYC, MYCL or MYCN). One of them is amplified and overexpressed in a mutually exclusive manner and represents the most prominent activating oncogene alteration in this malignancy. In particular, MYCN amplification is associated with tumor progression, treatment failure and poor prognosis. Given the role of MYCN in SCLC and its restricted expression profile, MYCN represents a promising therapeutic target; although it is considered undruggable by traditional approaches. An innovative approach to target the oncogene concerns specific MYCN expression inhibition, acting directly at the level of DNA, through an antigene peptide nucleic acid (agPNA) oligonucleotide, called BGA002. This thesis focused on the study of BGA002, as a possible targeted therapeutic strategy for the treatment of MYCN-related SCLC. In this context, BGA002 proved to be a specific and highly effective inhibitor. Furthermore, MYCN silencing induced alterations in many downstream pathways and led to apoptosis, in concomitance with autophagy reactivation. Moreover, systemic administration of BGA002 was effective in vivo as well, significantly increasing survival in MNA mouse models, even in the scenario of multidrug-resistance. In addition, BGA002 treatment successfully reduced N-Myc protein expression and, more importantly, caused a massive diminishment in tumor vascularization in the multidrug-resistant model. Overall, these results proved that MYCN inhibition by BGA002 may represent a new promising precision medicine approach, to treat MYCN-related SCLC.

Hetero-bifunctional molecules as possible therapeutics for the treatment of Alzheimer’s disease.

Alzheimer’s disease (AD) is the most common form of dementia, currently affecting more than 50 million people worldwide. In recent years attention towards this disease has risen in search for discovery and development of a drug that can stop it. Indeed, therapies for AD provide only temporary symptomatic relief. The cause for the high attrition rate for AD drug discovery has been attributed to several factors, including the fact that the AD pathogenesis is not yet fully understood. Nevertheless, what is increasingly recognized is that AD is a multifactorial syndrome, characterized by many conditions which may lead to neuronal death. Given this, it is widely accepted that a molecule able to modulate more than one target would bring benefit to the therapy of AD. In the first chapter of this thesis, there are reported two projects regarding the design and synthesis of new series of GSK-3/HDAC dual inhibitors, two of the main enzymes involved in AD. Two different series of compounds were synthesized and evaluated for their inhibitory activity towards the target enzymes. The best compounds of the series were selected for further biologic investigation to evaluate their properties. The second project focused on the design of non ATP-competitive GSK-3 inhibitors combined with HDAC inhibition properties. Also in this case, the best compounds of the series were selected for biologic investigation to further evaluate their properties. In chapter 2, the design and synthesis of a GSK-3-directed Proteolysis Targeting Chimeras (PROTAC), a new technology in drug discovery that act through degradation rather than inhibition, is reported. The design and synthesis of a small series of GSK-3-directed PROTACs was achieved. In vitro assays were performed to evaluate the GSK-3-degradation ability, the effective involvement of E3 ubiquitine ligase in the process and their neuroprotective abilities.

Molecular engineering of the m13 phage for targeted photodynamic cancer treatment

This thesis explores the advancement of cancer treatment through targeted photodynamic therapy (PDT) using bioengineered phages. It aims to harness the specificity of phages for targeting cancer-related receptors such as EGFR and HER2, which are pivotal in numerous malignancies and associated with poor outcomes. The study commenced with the M13EGFR phage, modified to target EGFR through pIII-displayed EGFR-binding peptides, demonstrating enhanced killing efficiency when conjugated with the Rose Bengal photosensitizer. This phase underscored phages' potential in targeted PDT. A breakthrough was achieved with the development of the M137D12 phage, engineered to display the 7D12 nanobody for precise EGFR targeting, marking a shift from peptide-based to nanobody-based targeting and yielding better specificity and therapeutic results. The translational potential was highlighted through in vitro and in vivo assays employing therapeutic lasers, showing effective, specific cancer cell killing through a necrotic mechanism. Additionally, the research delved into the interaction between the M13CC phage and colon cancer models, demonstrating its ability to penetrate and disrupt cancer spheroids only upon irradiation, indicating a significant advancement in targeting cells within challenging tumor microenvironments. In summary, the thesis provides a thorough examination of the phage platform's efficacy and versatility for targeted PDT. The promising outcomes, especially with the M137D12 phage, and initial findings on a HER2-targeting phage (M13HER2), forecast a promising future for phage-mediated, targeted anticancer strategies employing photosensitizers in PDT.

Minimal Invasive Ostheosintesis For Treatment Of Diaphiseal Transverse Humeral Shaft Fractures.

To evaluate patients with transverse fractures of the shaft of the humerus treated with indirect reduction and internal fixation with plate and screws through minimally invasive technique. Inclusion criteria were adult patients with transverse diaphyseal fractures of the humerus closed, isolated or not occurring within 15 days of the initial trauma. Exclusion criteria were patients with compound fractures. In two patients, proximal screw loosening occurred, however, the fractures consolidated in the same mean time as the rest of the series. Consolidation with up to 5 degrees of varus occurred in five cases and extension deficit was observed in the patient with olecranon fracture treated with tension band, which was not considered as a complication. There was no recurrence of infection or iatrogenic radial nerve injury. It can be concluded that minimally invasive osteosynthesis with bridge plate can be considered a safe and effective option for the treatment of transverse fractures of the humeral shaft. Level of Evidence III, Therapeutic Study.

The Effect Of Pelvic Floor Muscle Training Alone Or In Combination With Electrostimulation In The Treatment Of Sexual Dysfunction In Women With Multiple Sclerosis.

Sexual dysfunction (SD) affects up to 80% of multiple sclerosis (MS) patients and pelvic floor muscles (PFMs) play an important role in the sexual function of these patients. The objective of this paper is to evaluate the impact of a rehabilitation program to treat lower urinary tract symptoms on SD of women with MS. Thirty MS women were randomly allocated to one of three groups: pelvic floor muscle training (PFMT) with electromyographic (EMG) biofeedback and sham neuromuscular electrostimulation (NMES) (Group I), PFMT with EMG biofeedback and intravaginal NMES (Group II), and PFMT with EMG biofeedback and transcutaneous tibial nerve stimulation (TTNS) (Group III). Assessments, before and after the treatment, included: PFM function, PFM tone, flexibility of the vaginal opening and ability to relax the PFMs, and the Female Sexual Function Index (FSFI) questionnaire. After treatment, all groups showed improvements in all domains of the PERFECT scheme. PFM tone and flexibility of the vaginal opening was lower after the intervention only for Group II. All groups improved in arousal, lubrication, satisfaction and total score domains of the FSFI questionnaire. This study indicates that PFMT alone or in combination with intravaginal NMES or TTNS contributes to the improvement of SD.

Impact Of Benign Prostatic Hyperplasia Pharmacological Treatment On Transrectal Prostate Biopsy Adverse Effects.

Background. Benign prostatic hyperplasia (BPH) pharmacological treatment may promote a decrease in prostate vascularization and bladder neck relaxation with theoretical improvement in prostate biopsy morbidity, though never explored in the literature. Methods. Among 242 consecutive unselected patients who underwent prostate biopsy, after excluding those with history of prostate biopsy/surgery or using medications not for BPH, we studied 190 patients. On the 15th day after procedure patients were questioned about symptoms lasting over a week and classified according to pharmacological BPH treatment. Results. Thirty-three patients (17%) were using alpha-blocker exclusively, five (3%) 5-alpha-reductase inhibitor exclusively, twelve (6%) patients used both medications, and 140 (74%) patients used none. There was no difference in regard to age among groups (P = 0.5). Postbiopsy adverse effects occurred as follows: hematuria 96 (50%), hematospermia 53 (28%), hematochezia 22 (12%), urethrorrhagia 19 (10%), fever 5 (3%), and pain 20 (10%). There was a significant negative correlation between postbiopsy hematuria and BPH pharmacological treatment with stronger correlation for combined use of 5-alpha-reductase inhibitor and alpha-blocker over 6 months (P = 0.0027). Conclusion. BPH pharmacological treatment, mainly combined for at least 6 months seems to protect against prostate biopsy adverse effects. Future studies are necessary to confirm our novel results.

Evaluation Of Different Treatment Methods Against Denture Stomatitis: A Randomized Clinical Study.

The aim of this clinical study was to determine the efficacy of Uncaria tomentosa (cat's claw) against denture stomatitis (DS). Fifty patients with DS were randomly assigned into 3 groups to receive 2% miconazole, placebo, or 2% U tomentosa gel. DS level was recorded immediately, after 1 week of treatment, and 1 week after treatment. The clinical effectiveness of each treatment was measured using Newton's criteria. Mycologic samples from palatal mucosa and prosthesis were obtained to determinate colony forming units per milliliter (CFU/mL) and fungal identification at each evaluation period. Candida species were identified with HiCrome Candida and API 20C AUX biochemical test. DS severity decreased in all groups (P < .05). A significant reduction in number of CFU/mL after 1 week (P < .05) was observed for all groups and remained after 14 days (P > .05). C albicans was the most prevalent microorganism before treatment, followed by C tropicalis, C glabrata, and C krusei, regardless of the group and time evaluated. U tomentosa gel had the same effect as 2% miconazole gel. U tomentosa gel is an effective topical adjuvant treatment for denture stomatitis.

Aquatic Toxicity Of Dyes Before And After Photo-fenton Treatment.

This study evaluated the ecotoxicity of five dyes to freshwater organisms before and during their photo-Fenton degradation. EC50 (48h) of the five tested dyes ranged from of 6.9 to >1000mgL(-1) for Daphnia similis. In the chronic tests IC50 (72h) varied from 65 to >100mgL(-1) for Pseudokirchneriella subcapitata and IC50 (8 days) from 0.5 to 410mgL(-1) for Ceriodaphnia dubia. Toxicity tests revealed that although the applied treatment was effective for decolorization of the dye, the partial mineralization may be responsible for the presence of degradation products which can be either more toxic than the original dye, as is the case of Vat Green 3 and Reactive Black 5, lead to initially toxic products which may be further degraded to non toxic products (acid Orange 7 and Food Red 17), or generate non toxic products as in the case of Food Yellow 3. The results highlighted the importance of assessing both acute and chronic toxicity tests of treated sample before effluent discharge.

The Consequences Of Refractory Epilepsy And Its Treatment.

Seizures in some 30% to 40% of patients with epilepsy fail to respond to antiepileptic drugs or other treatments. While much has been made of the risks of new drug therapies, not enough attention has been given to the risks of uncontrolled and progressive epilepsy. This critical review summarizes known risks associated with refractory epilepsy, provides practical clinical recommendations, and indicates areas for future research. Eight international epilepsy experts from Europe, the United States, and South America met on May 4, 2013, to present, review, and discuss relevant concepts, data, and literature on the consequences of refractory epilepsy. While patients with refractory epilepsy represent the minority of the population with epilepsy, they require the overwhelming majority of time, effort, and focus from treating physicians. They also represent the greatest economic and psychosocial burdens. Diagnostic procedures and medical/surgical treatments are not without risks. Overlooked, however, is that these risks are usually smaller than the risks of long-term, uncontrolled seizures. Refractory epilepsy may be progressive, carrying risks of structural damage to the brain and nervous system, comorbidities (osteoporosis, fractures), and increased mortality (from suicide, accidents, sudden unexpected death in epilepsy, pneumonia, vascular disease), as well as psychological (depression, anxiety), educational, social (stigma, driving), and vocational consequences. Adding to this burden is neuropsychiatric impairment caused by underlying epileptogenic processes (essential comorbidities), which appears to be independent of the effects of ongoing seizures themselves. Tolerating persistent seizures or chronic medicinal adverse effects has risks and consequences that often outweigh risks of seemingly more aggressive treatments. Future research should focus not only on controlling seizures but also on preventing these consequences.