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Delivery represents a major hurdle to the clinical advancement of oligonucleotide therapeutics for the treatment of disorders such as Duchenne muscular dystrophy (DMD). In this preliminary study, we explored the ability of 2'-O-methyl-phosphorothioate antisense oligonucleotides (ASOs) conjugated with lipophilic ursodeoxycholic acid (UDCA) to permeate across intestinal barriers in vitro by a co-culture system of non-contacting IEC-6 cells and DMD myotubes, either alone or encapsulated in exosomes. UDCA was used to enhance the lipophilicity and membrane permeability of ASOs, potentially improving oral bioavailability. Exosomes were employed due to their biocompatibility and ability to deliver therapeutic cargo across biological barriers. Exon skipping was evaluated in the DMD myotubes to reveal the targeting efficiency. Exosomes extracted from milk and wild-type myotubes loaded with 5'-UDC-3'Cy3-ASO and seeded directly on DMD myotubes appear able to fuse to myotubes and induce exon skipping, up to ~20%. Permeation studies using the co-culture system were performed with 5'-UDC-3'Cy3-ASO 51 alone or loaded in milk-derived exosomes. In this setting, only gymnotic delivery induced significant levels of exon skipping (almost 30%) implying a possible role of the intestinal cells in enhancing delivery of ASOs. These results warrant further investigations to elucidate the delivery of ASOs by gymnosis or exosomes.
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Herein, we report the synthesis of a new hybrid compound based on a 2'-deoxyuridine nucleoside conjugated with a NO photo-donor moiety (dU-t-NO) via CuAAC click chemistry. Hybrid dU-t-NO, as well as two previously reported 2'-deoxyadenosine based hybrids (dAdo-S-NO and dAdo-t-NO), were evaluated for their cytotoxic and cytostatic activities in selected cancer cell lines. dAdo-S-NO and dAdo-t-NO hybrids displayed higher activity with respect to dU-t-NO. All hybrids showed effective release of NO in the micromolar range. The photochemical behavior of the newly reported hybrid, dU-t-NO, was studied in the RKO colon carcinoma cell line, whereas the dAdo-t-NO hybrid was tested in both colon carcinoma RKO and hepatocarcinoma Hep 3B2.1-7 cell lines to evaluate the potential effect of NO released upon irradiation on cell viability. A customized irradiation apparatus for in vitro experiments was also designed.
Assuntos
Antineoplásicos , Doadores de Óxido Nítrico , Óxido Nítrico , Nucleosídeos , Humanos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Óxido Nítrico/metabolismo , Óxido Nítrico/química , Doadores de Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/química , Nucleosídeos/química , Nucleosídeos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Química Click , Proliferação de Células/efeitos dos fármacos , Estrutura Molecular , Desoxiuridina/química , Desoxiuridina/farmacologia , Desoxiuridina/análogos & derivadosRESUMO
Ursodeoxycholic acid (UDCA) and acetoacetate are natural compounds present in the human intestine and blood, respectively. A number of studies highlighted that besides their well-known primary biological roles, both compounds possess the ability to influence a variety of cellular processes involved in the etiology of various diseases. These reasons suggested the potential of acetoacetate-UDCA hybrids as possible therapeutic agents and prompted us to develop a synthetic strategy to selectively derivatize the hydroxyl groups of the bile acid with acetoacetyl moieties. 3α-acetoacetoxy UDCA was obtained (60% isolated yield) via the regioselective transesterification of methyl acetoacetate with UDCA promoted by the Candida antarctica lipase B (CAL-B). 3α,7ß-bis-acetoacetoxy UDCA was obtained instead by thermal condensation of methyl acetoacetate and UDCA (80% isolated yield). This bis-adduct was finally converted to the 7ß-acetoacetoxy UDCA (82% isolated yield) via CAL-B catalyzed regioselective alcoholysis of the ester group on the 3α position. In order to demonstrate the value of the above new hybrids as UDCA-based scaffolds, 3α-acetoacetoxy UDCA was subjected to multicomponent Biginelli reaction with benzaldehyde and urea to obtain the corresponding 4-phenyl-3,4-dihydropyrimidin-2-(1H)-one derivative in 65% isolated yield.
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Acetoacetatos , Ácido Ursodesoxicólico , Humanos , Ácidos e Sais BiliaresRESUMO
Here, we propose the molecular hybridization of dihydroartemisinin (DHA) and ursodeoxycholic bile acid (UDCA), approved drugs, for the preparation of antiviral agents against SARS-CoV-2. DHA and UDCA were selected on the basis of their recently demonstrated in vitro activity against SARS-CoV-2. A selection of DHA-UDCA-based hybrids obtained by varying the nature of the linkage and the bile acid conjugation point as well as unconjugated DHA and UDCA were tested in vitro for cytotoxicity and anti-SARS-CoV-2 activity on Vero E6 and Calu-3 human lung cells. The hybrid DHA-t-UDCMe, obtained by conjugation via click chemistry on a gram scale, was identified as a potential candidate for SARS-CoV-2 infection treatment due to significant reduction of viral replication, possibly involving ACE2 downregulation, no cytotoxicity, and chemical stability.
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Artemisinin is a natural compound extracted from Artemisia species belonging to the Asteraceae family. Currently, artemisinin and its derivatives are considered among the most significant small-molecule antimalarial drugs. Artemisinin and its derivatives have also been shown to possess selective anticancer properties, however, there are several limitations and gaps in knowledge that retard their repurposing as effective anticancer agents. Hybridization resulting from a covalent combination of artemisinin with one or more active pharmacophores has emerged as a promising approach to overcome several issues. The variety of hybridization partners allows improvement in artemisinin activity by tuning the ability of conjugated artemisinin to interact with various molecule targets involved in multiple biological pathways. This review highlights the current scenario of artemisinin-derived hybrids with potential anticancer activity. The synthetic approaches to achieve the corresponding hybrids and the structure-activity relationships are discussed to facilitate further rational design of more effective candidates.
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Hepatocellular carcinoma is the third most common cause of cancer-related death according to the International Agency for Research on Cancer. Dihydroartemisinin (DHA), an antimalarial drug, has been reported to exhibit anticancer activity but with a short half-life. We synthesized a series of bile acid-dihydroartemisinin hybrids to improve its stability and anticancer activity and demonstrated that an ursodeoxycholic-DHA (UDC-DHA) hybrid was 10-fold more potent than DHA against HepG2 hepatocellular carcinoma cells. The objectives of this study were to evaluate the anticancer activity and investigate the molecular mechanisms of UDCMe-Z-DHA, a hybrid of ursodeoxycholic acid methyl ester and DHA via a triazole linkage. We found that UDCMe-Z-DHA was even more potent than UDC-DHA in HepG2 cells with IC50 of 1 µM. Time course experiments and stability in medium determined by cell viability assay as well as HPLC-MS/MS analysis revealed that UDCMe-Z-DHA was more stable than DHA, which in part accounted for the increased anticancer activity. Mechanistic studies revealed that UDCMe-Z-DHA caused G0/G1 arrest and induced reactive oxygen species (ROS), mitochondrial membrane potential loss and autophagy, which may in turn lead to apoptosis. Compared to DHA, UDCMe-Z-DHA displayed much lower cytotoxicity toward normal cells. Thus, UDCMe-Z-DHA may be a potential drug candidate for hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Ácido Ursodesoxicólico , Neoplasias Hepáticas/patologia , Espectrometria de Massas em Tandem , Apoptose , Artemeter , Linhagem Celular TumoralRESUMO
INTRODUCTION: Pediatric-onset multiple sclerosis (POMS) is characterized by high inflammatory disease activity. Our aim was to describe the treatment sequencing and report the impact highly effective disease-modifying treatment (HET) had on disease activity. MATERIALS AND METHODS: Five consecutive patients with POMS were administered HET following lower efficacy drug or as initial therapy. Data on treatment sequencing, relapses and MRIs were collected during the follow-up. RESULTS: Our patients had an average age of 13.8 years (range 9-17) at diagnosis and 13.4 years (range 9-16) at disease onset, and 2/5 (40%) POMS were female. The pre-treatment average annualized relapse rate was 1.6 (range 0.8-2.8), and the average follow-up length was 5 years (range 3-7). A total of 2/5 (40%) patients were stable on HET at initial therapy, and 3/5 (60%) required an escalation to more aggressive treatment, even if two of them had been put on HET as initial treatment. Four out of five patients (80%) had No Evidence of Disease Activity-3 status (NEDA-3) at an average follow-up of 3 years (range 2-5). CONCLUSION: It has been observed that in a recent time period all the cases had prompt diagnosis, early HET or escalation to HET with a good outcome in 80% of the cases.
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OBJECTIVES: To assess whether COVID-19 could be a concurrent factor in the genesis and/or worsening of stroke and to provide data on COVID-19 -associated stroke patients during the first pandemic wave and comparative data on COVID-19 negative stroke patients in the same period. MATERIALS AND METHODS: This is a retrospective, observational, case-control, single centre study, carried out in a General Hospital in northern Italy. Sixty-three consecutive stroke patients were included, COVID-19-associated stroke was classified as cases and non COVID-19-associated stroke as controls. RESULTS: A total of 19/63 (28.8%) had a COVID-19-associated stroke, 11 /63 (17.5%) were haemorrhagic and 52/63 (82.5%) ischaemic. COVID-19-associated strokes were more severe (p-value 0.019) and had a higher risk of severe disability and/or death (OR 3.79, CI 95%: 1.21-11.93, p-value 0.19). The COVID-19-associated stroke patients with onset during hospitalization for COVID-19 had a more severe stroke than patients with COVID-19 onset during hospitalization for stroke (p-value 0.019). CONCLUSION: Although no relationship was observed between the stroke aetiology and COVID-19, intriguingly, COVID-associated stroke turned out to be more severe and disabling. Hopefully, further studies will provide more data and help in the management of this emerging population.
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COVID-19 , Doenças Transmissíveis , Acidente Vascular Cerebral , Humanos , COVID-19/complicações , COVID-19/diagnóstico , SARS-CoV-2 , Estudos de Casos e Controles , Pandemias , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/terapia , Acidente Vascular Cerebral/complicações , Estudos Retrospectivos , Doenças Transmissíveis/complicaçõesRESUMO
The COVID-19 pandemic poses an ongoing global challenge, and several risk factors make people with multiple sclerosis (pwMS) particularly susceptible to running a severe disease course. Although the literature does report numerous articles on the risk factors for severe COVID-19 and vaccination response in pwMS, there is a scarcity of reviews integrating both these aspects into strategies aimed at minimizing risks. The aim of this review is to describe the risk of vulnerable pwMS exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the issues related to the SARS-CoV-2 vaccine and to evidence possible future strategies in the clinical management of pwMS. The authors searched for papers on severe COVID-19 risk factors, SARS-CoV-2 vaccination and people with multiple sclerosis in support of this narrative literature review. We propose a multilevel strategy aimed at: the evaluation of risk factors for severe COVID-19 in people with multiple sclerosis, identifying the most appropriate vaccination schedule that is safe for people on disease-modifying drugs (DMDs) and a strict follow-up of high-risk people with multiple sclerosis to allow for the prompt administration of monoclonal antibodies to manage COVID-19 risks in this patient population.
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Our groups previously reported that conjugation at 3'-end with ursodeoxycholic acid (UDCA) significantly enhanced in vitro exon skipping properties of ASO 51 oligonucleotide targeting the human DMD exon 51. In this study, we designed a series of lipophilic conjugates of ASO 51, to explore the influence of the lipophilic moiety on exon skipping efficiency. To this end, three bile acids and two fatty acids have been derivatized and/or modified and conjugated to ASO 51 by automatized solid phase synthesis. We measured the melting temperature (Tm) of lipophilic conjugates to evaluate their ability to form a stable duplex with the target RNA. The exon skipping efficiency has been evaluated in myogenic cell lines first in presence of a transfection agent, then in gymnotic conditions on a selection of conjugated ASO 51. In the case of 5'-UDC-ASO 51, we also evaluated the influence of PS content on exon skipping efficiency; we found that it performed better exon skipping with full PS linkages. The more efficient compounds in terms of exon skipping were found to be 5'-UDC- and 5',3'-bis-UDC-ASO 51.
Assuntos
Distrofia Muscular de Duchenne , Linhagem Celular , Distrofina/genética , Éxons/genética , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/genéticaRESUMO
Chenodeoxycholic acid and ursodeoxycholic acid (CDCA and UDCA, respectively) have been conjugated with paclitaxel (PTX) anticancer drugs through a high-yield condensation reaction. Bile acid-PTX hybrids (BA-PTX) have been investigated for their pro-apoptotic activity towards a selection of cancer cell lines as well as healthy fibroblast cells. Chenodeoxycholic-PTX hybrid (CDC-PTX) displayed cytotoxicity and cytoselectivity similar to PTX, whereas ursodeoxycholic-PTX hybrid (UDC-PTX) displayed some anticancer activity only towards HCT116 colon carcinoma cells. Pacific Blue (PB) conjugated derivatives of CDC-PTX and UDC-PTX (CDC-PTX-PB and UDC-PTX-PB, respectively) were also prepared via a multistep synthesis for evaluating their ability to enter tumor cells. CDC-PTX-PB and UDC-PTX-PB flow cytometry clearly showed that both CDCA and UDCA conjugation to PTX improved its incoming into HCT116 cells, allowing the derivatives to enter the cells up to 99.9%, respect to 35% in the case of PTX. Mean fluorescence intensity analysis of cell populations treated with CDC-PTX-PB and UDC-PTX-PB also suggested that CDC-PTX-PB could have a greater ability to pass the plasmatic membrane than UDC-PTX-PB. Both hybrids showed significant lower toxicity with respect to PTX on the NIH-3T3 cell line.
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Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/farmacologia , Paclitaxel/química , Paclitaxel/farmacologia , Animais , Antineoplásicos Fitogênicos/síntese química , Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/síntese química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Ácido Desoxicólico/análogos & derivados , Ácido Desoxicólico/síntese química , Ácido Desoxicólico/química , Ácido Desoxicólico/farmacologia , Humanos , Leucemia/tratamento farmacológico , Camundongos , Paclitaxel/análogos & derivados , Paclitaxel/síntese químicaRESUMO
Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2'-OMe PS oligonucleotide conjugated at 3'-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3'-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3'-UDC with an average 9.5-fold increase with respect to ASO 51.
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Éxons , Distrofia Muscular de Duchenne , Mioblastos Esqueléticos/metabolismo , Oligonucleotídeos Antissenso , Precursores de RNA , Ácido Ursodesoxicólico , Linhagem Celular Transformada , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/farmacologia , Precursores de RNA/genética , Precursores de RNA/metabolismo , Ácido Ursodesoxicólico/química , Ácido Ursodesoxicólico/farmacocinética , Ácido Ursodesoxicólico/farmacologiaRESUMO
A 9-year-old neutered male Dachshund dog was assessed for stranguria. An enlarged prostate was identified on physical examination, and a diagnosis of prostatic carcinoma confirmed by cytology. Due to a neoplastic lower urinary tract obstruction, palliative surgical urinary diversion treatment was performed with laparoscopic assisted cutaneous ureterostomy (LACU). The dog recovered well without any major complications. Adjuvant chemotherapy was maintained and continued for post-surgical medical therapy. This report describes a novel minimally invasive assisted technique in canine patients for palliative treatment of prostatic neoplasia.
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Carcinoma , Doenças do Cão , Laparoscopia , Derivação Urinária , Animais , Carcinoma/veterinária , Doenças do Cão/cirurgia , Cães , Laparoscopia/veterinária , Masculino , Ureterostomia/métodos , Ureterostomia/veterinária , Derivação Urinária/métodos , Derivação Urinária/veterináriaRESUMO
The click azide = alkyne 1,3-dipolar cycloaddition (click chemistry) has become the approach of choice for bioconjugations in medicinal chemistry, providing facile reaction conditions amenable to both small and biological molecules. Many nucleoside analogs are known for their marked impact in cancer therapy and for the treatment of virus diseases and new targeted oligonucleotides have been developed for different purposes. The click chemistry allowing the tolerated union between units with a wide diversity of functional groups represents a robust means of designing new hybrid compounds with an extraordinary diversity of applications. This review provides an overview of the most recent works related to the use of click chemistry methodology in the field of nucleosides, nucleotides and nucleic acids for pharmacological applications.
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Alcinos/química , Azidas/química , Ácidos Nucleicos/química , Nucleosídeos/química , Nucleotídeos/química , Adenosina/química , Animais , Linhagem Celular Tumoral , Química Click , Reação de Cicloadição , DNA/química , Receptores ErbB/química , Humanos , Camundongos , Oligonucleotídeos Antissenso/química , RNA Guia de Cinetoplastídeos/química , Reprodutibilidade dos Testes , Tecnologia Farmacêutica , Triazóis/químicaRESUMO
BACKGROUND AND PURPOSE: an estimated 40-80% of acute ischemic stroke patients have dysphagia and about 14% develop stroke-associated pneumonia. However, it may be difficult to detect swallowing problems at admission. Moreover, there might not be an on-duty specialist skilled in the diagnosis of this condition. This study aimed at developing a user-friendly bedside examination to identify the risk of dysphagia in stroke patients at hospital admission. METHODS: a diagnostic accuracy study was carried out to assess the concurrent validity of a simple Bedside Screening Tool for Dysphagia (BSTD) in acute stroke. All the consecutive stroke patients admitted between January and April 2018 were enrolled. Sensitivity, specificity, positive (PPV), negative predictive values (NPV) and the Cohen K concordance index scores, reported by nurses and speech-pathologists, were assessed. RESULTS: a total of 67/120 patients (55.8%) were male; overall average age was 67.4 (range 45-91) and 80.8% of the whole population had a history of ischemic stroke. The nursing staff identified 33.3% of dysphagia cases at admission and the speech pathologists 30%. The Cohen K was 0.92 (optimal concordance when K was > 0.8), sensitivity was 100%, specificity 95.2%, PPV 90% and NPV 100%. CONCLUSIONS: our BSTD had a 100% negative predictive value, indicating that this screening test is very useful in ruling out/confirming dysphagia in acute stroke patients.
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Transtornos de Deglutição/diagnóstico , Deglutição , Testes Imediatos , Acidente Vascular Cerebral/complicações , Idoso , Idoso de 80 Anos ou mais , Transtornos de Deglutição/etiologia , Transtornos de Deglutição/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Reprodutibilidade dos Testes , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in adults and accounts for 85-90% of all primary liver cancer. Based on the estimation by the International Agency for Research on Cancer in 2018, liver cancer is the fourth leading cause of cancer death globally. Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is a well-known drug for the treatment of malaria. Previous studies have demonstrated that DHA exhibits antitumor effects toward a variety of human cancers and has a potential for repurposing as an anticancer drug. However, its short half-life is a concern and may limit the application in cancer therapy. We have reported that UDC-DHA, a hybrid of bile acid ursodeoxycholic acid (UDCA) and DHA, is â¼12 times more potent than DHA against a HCC cell line HepG2. In this study, we found that UDC-DHA was also effective against another HCC cell line Huh-7 with an IC50 of 2.16 µM, which was 18.5-fold better than DHA with an IC50 of 39.96 µM. UDC-DHA was much more potent than the combination of DHA and UDCA at 1:1 molar ratio, suggesting that the covalent linkage rather than a synergism between UDCA and DHA is critical for enhancing DHA potency in HepG2 cells. Importantly, UDC-DHA was much less toxic to normal cells than DHA. UDC-DHA induced G0/G1 arrest and apoptosis. Both DHA and UDC-DHA significantly elevated cellular reactive oxygen species generation but with different magnitude and timing in HepG2 cells; whereas only DHA but not UDC-DHA induced reactive oxygen species in Huh-7 cells. Depolarization of mitochondrial membrane potential was detected in both HepG2 and Huh-7 cells and may contribute to the anticancer effect of DHA and UDC-DHA. Furthermore, UDC-DHA was much more stable than DHA based on activity assays and high performance liquid chromatography-MS/MS analysis. In conclusion, UDC-DHA and DHA may exert anticancer actions via similar mechanisms but a much lower concentration of UDC-DHA was required, which could be attributed to a better stability of UDC-DHA. Thus, UDC-DHA could be a better drug candidate than DHA against HCC and further investigation is warranted.
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The advantages of a treatment modality that combines two or more therapeutic agents in cancer therapy encourages the study of hybrid functional compounds for pharmacological applications. In light of this, we reviewed recent works on hybrid molecules based on bile acids. Due to their biological properties, as well as their different chemical/biochemical reactive moieties, bile acids can be considered very interesting starting molecules for conjugation with natural or synthetic bioactive molecules.
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Antineoplásicos/farmacologia , Ácidos e Sais Biliares/farmacologia , Neoplasias/tratamento farmacológico , Animais , HumanosAssuntos
Betacoronavirus , Infecções por Coronavirus/complicações , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/complicações , Pneumonia Viral/diagnóstico , Acidente Vascular Cerebral/etiologia , Idoso , Idoso de 80 Anos ou mais , COVID-19 , Estudos de Coortes , Infecções por Coronavirus/terapia , Feminino , Humanos , Itália , Masculino , Pandemias , Pneumonia Viral/etiologia , Pneumonia Viral/terapia , SARS-CoV-2 , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/terapiaRESUMO
Oligonucleotides (ONs) are gaining increasing importance as a promising novel class of biopharmaceuticals. Thanks to their fundamental role in gene regulation, they can be used to develop custom-made drugs (also called N-to-1) able to act on the gene expression at pre-translational level. With recent approvals of ON-based therapeutics by the Food and Drug Administration (FDA), a growing demand for high-quality chemically modified ONs is emerging and their market is expected to impressively prosper in the near future. To satisfy this growing market demand, a scalable and economically sustainable ON production is needed. In this paper, the state of the art of the whole ON production process is illustrated with the aim of highlighting the most promising routes toward the auspicated market-size production. In particular, the most recent advancements in both the upstream stage, mainly based on solid-phase synthesis and recombinant technology, and the downstream one, focusing on chromatographic techniques, are reviewed. Since ON production is projected to expand to the large scale, automatized multicolumn countercurrent technologies will reasonably be required soon to replace the current ones based on batch single-column operations. This consideration is supported by a recent cutting-edge application of continuous chromatography for the ON purification.