Exploring treatment strategies for paroxysmal nocturnal hemoglobinuria: an overview of registered clinical trials.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disease in which blood cells lack anchored proteins that regulate the complement system. The erythrocytes are then destroyed because of uncontrolled complement activity, leading to intravascular hemolysis (IVH) and a high risk of thrombosis outcome. A huge alteration in the treatment of the disease was the development of terminal complement inhibitors, with the achievement of IVH blockade, reduction or abolishment of red blood cell (RBC) transfusions, and thromboembolic events prevention. However, patients treated with these inhibitors can still present extravascular hemolysis (EVH) caused by C3 activation and residual IVH or clinically relevant levels of breakthrough hemolysis (BTH). Proximal complement inhibitors turned out to be the key to the solution of this problem by targeting components of the proximal complement pathway, avoiding intra and extravascular hemolysis. FDA approved eculizumab, ravulizumab (terminal inhibitors), pegcetacoplan, iptacopan, and danicopan (proximal inhibitors) as a treatment for PNH so far. Various clinical trials are underway to find the most effective method to treat patients with PNH. This review aimed to summarize 71 registered clinical trials in the ClinicalTrials.gov database with the various treatment drugs, possible mechanisms, and novel findings related to PNH treatment.

From the Gut to the Brain: Is Microbiota a New Paradigm in Parkinson's Disease Treatment?

Parkinson's disease (PD) is recognized as the second most prevalent primary chronic neurodegenerative disorder of the central nervous system. Clinically, PD is characterized as a movement disorder, exhibiting an incidence and mortality rate that is increasing faster than any other neurological condition. In recent years, there has been a growing interest concerning the role of the gut microbiota in the etiology and pathophysiology of PD. The establishment of a brain-gut microbiota axis is now real, with evidence denoting a bidirectional communication between the brain and the gut microbiota through metabolic, immune, neuronal, and endocrine mechanisms and pathways. Among these, the vagus nerve represents the most direct form of communication between the brain and the gut. Given the potential interactions between bacteria and drugs, it has been observed that the therapies for PD can have an impact on the composition of the microbiota. Therefore, in the scope of the present review, we will discuss the current understanding of gut microbiota on PD and whether this may be a new paradigm for treating this devastating disease.

Evaluation of the impact of two C5 genetic variants on C5-eculizumab complex stability at the molecular level.

Complement C5 is the target of the monoclonal antibody eculizumab, used in complement dysregulating disorders, like the rare disease Paroxysmal Nocturnal Hemoglobinuria (PNH). PNH is an acquired hematopoietic stem cell condition characterized by aberrant destruction of erythrocytes, chronic hemolytic anemia, and thromboembolism propensity. C5 is a protein component of the complement system which is part of the immune system of the body and plays a prominent role in the destruction of red blood cells, misidentifying them as a threat. This work describes the application of molecular dynamics simulations to the study of the underlying interactions between complement C5 and eculizumab. This study also reveals the importance of single nucleotide polymorphisms on C5 protein concerning the effective inhibition of the mAB, involving the mechanistic events taking place at the interface spots of the complex. The predicted conformational change in the C5 Arg885/His/Cys mutation has implications on the protein's interaction with eculizumab, compromising their compatibility. The acquired insights into the conformational changes, dynamics, flexibility, and interactions shed light on the knowledge of the function of this biomolecule providing answers about the poor response to the treatment in PNH patient carriers of the mutations. By investigating the intricate dynamics, significant connections between C5 and eculizumab can be uncovered. Such insights may aid in the creation of novel compounds or lead to the enhancement of eculizumab's efficacy.Communicated by Ramaswamy H. Sarma.

Betulinic Acid for Glioblastoma Treatment: Reality, Challenges and Perspectives.

Betulinic acid is a naturally occurring compound that can be obtained through methanolic or ethanolic extraction from plant sources, as well as through chemical synthesis or microbial biotransformation. Betulinic acid has been investigated for its potential therapeutic properties, and exhibits anti-inflammatory, antiviral, antimalarial, and antioxidant activities. Notably, its ability to cross the blood-brain barrier addresses a significant challenge in treating neurological pathologies. This review aims to compile information about the impact of betulinic acid as an antitumor agent, particularly in the context of glioblastoma. Importantly, betulinic acid demonstrates selective antitumor activity against glioblastoma cells by inhibiting proliferation and inducing apoptosis, consistent with observations in other cancer types. Compelling evidence published highlights the acid's therapeutic action in suppressing the Akt/NFκB-p65 signaling cascade and enhancing the cytotoxic effects of the chemotherapeutic agent temozolomide. Interesting findings with betulinic acid also suggest a focus on researching the reduction of glioblastoma's invasiveness and aggressiveness profile. This involves modulation of extracellular matrix components, remodeling of the cytoskeleton, and secretion of proteolytic proteins. Drawing from a comprehensive review, we conclude that betulinic acid formulations as nanoparticles and/or ionic liquids are promising drug delivery approaches with the potential for translation into clinical applications for the treatment and management of glioblastoma.

Noradrenergic Pathways Involved in Micturition in an Animal Model of Hydrocephalus-Implications for Urinary Dysfunction.

Hydrocephalus is characterized by enlargement of the cerebral ventricles, accompanied by distortion of the periventricular tissue. Patients with hydrocephalus usually experience urinary impairments. Although the underlying etiology is not fully described, the effects of hydrocephalus in the neuronal network responsible for the control of urination, which involves periventricular areas, including the periaqueductal gray (PAG) and the noradrenergic locus coeruleus (LC). In this study, we aimed to investigate the mechanisms behind urinary dysfunction in rats with kaolin-induced hydrocephalus. For that purpose, we used a validated model of hydrocephalus-the rat injected with kaolin in the cisterna magna-also presents urinary impairments in order to investigate the putative involvement of noradrenergic control from the brain to the spinal cord Onuf's nucleus, a key area in the motor control of micturition. We first evaluated bladder contraction capacity using cystometry. Since our previous characterization of the LC in hydrocephalic animals showed increased levels of noradrenaline, we then evaluated the noradrenergic innervation of the spinal cord's Onuf's nucleus by measuring levels of dopamine ß-hydroxylase (DBH). We also evaluated the expression of the c-Fos protooncogene, the most widely used marker of neuronal activation, in the ventrolateral PAG (vlPAG), an area that plays a major role in the control of urination by its indirect control of the LC via pontine micturition center. Hydrocephalic rats showed an increased frequency of bladder contractions and lower minimum pressure. These animals also presented increased DBH levels at the Onuf´s nucleus, along with decreased c-Fos expression in the vlPAG. The present findings suggest that impairments in urinary function during hydrocephalus may be due to alterations in descending noradrenergic modulation. We propose that the effects of hydrocephalus in the decrease of vlPAG neuronal activation lead to a decrease in the control over the LC. The increased availability of noradrenaline production at the LC probably causes an exaggerated micturition reflex due to the increased innervation of the Onuf´s nucleus, accounting for the urinary impairments detected in hydrocephalic animals. The results of the study provide new insights into the neuronal underlying mechanisms of urinary dysfunction in hydrocephalus. Further research is needed to fully evaluate the translational perspectives of the current findings.

Synthesis and Biological Evaluation of Amphotericin B Formulations Based on Organic Salts and Ionic Liquids against Leishmania infantum.

Nowadays, organic salts and ionic liquids (OSILs) containing active pharmaceutical ingredients (APIs) are being explored as drug delivery systems in modern therapies (OSILs-API). In that sense, this work is focused on the development of novel OSILs-API based on amphotericin B through an innovative procedure and the evaluation of the respective biological activity against Leishmania infantum. Several ammonium, methylimidazolium, pyridinium and phosphonium organic cations combined with amphotericin B as anion were synthesized in moderate to high yields and high purities by the water-reduced buffer neutralization method. All prepared compounds were characterized to confirm the desired chemical structure and the specific optical rotation ([α]D25) was also determined. The biological assays performed on L. infantum promastigotes showed increased activity against this parasitic disease when compared with the starting chloride forms and amphotericin B alone, highlighting [P6,6,6,14][AmB] as the most promising formulation. Possible synergism in the antiprotozoal activity was also evaluated for [P6,6,6,14][AmB], since it was proven to be the compound with the highest toxicity. This work reported a simple synthetic method, which can be applied to prepare other organic salts based on molecules containing fragile chemical groups, demonstrating the potential of these OSILs-AmB as possible agents against leishmaniasis.

Serine-based surfactants as effective antimicrobial agents against multiresistant bacteria.

The antimicrobial activity of two serine derived gemini cationic surfactants, amide (12Ser)2CON12 and ester (12Ser)2COO12, was tested using sensitive, E. coli ATCC 25922 and S. aureus ATCC 6538, and resistant, E. coli CTX M2, E. coli TEM CTX M9 and S. aureus ATCC 6538 and S. aureus MRSA ATCC 43300 Gram-positive and Gram-negative bacteria strains. Very low MIC values (5 µM) were found for the two resistant strains E.coli TEM CTX M9 and S. aureus MRSA ATCC 43300, in the case of the amide derivative, and for S. aureus MRSA ATCC 43300, in the case of the ester derivative. The interaction of the serine amphiphiles with lipid-model membranes (DPPG and DPPC) was investigated using Langmuir monolayers. A more pronounced effect on the DPPG than on the DPPC monolayer was observed. The effect induced by the surfactants on bacteria membrane was explored by Atomic Force Microscopy. A clear disruption of the bacteria membrane was observed for E. coli TEM CTX M9 upon treatment with (12ser)2CON12, whereas for the S. aureus MRSA few observable changes in cell morphology were found after treatment with either of the two surfactants. The cytotoxicity of the two compounds was assessed by hemolysis assay on human red blood cells (RBC). The compounds were shown to be non-cytotoxic up to 10 µM. Overall, the results reveal a promising potential, in particular of the amide derivative, as antimicrobial agent for two strains of antibiotic resistant bacteria.

Surfing the Third Wave of Ionic Liquids: A Brief Review on the Role of Surface-Active Ionic Liquids in Drug Development and Delivery.

The relevance of ionic liquids (ILs) is now well established in many fields, as their unique properties make them appealing as 1) greener alternatives to organic solvents (first-generation ILs), 2) tunable task-specific materials (second-generation ILs), and 3) multifunctional players in life and pharmaceutical sciences (third-generation ILs). This third wave of ILs encompasses a wide range of compounds, from bioactive molecules with single or even dual therapeutic action, to potential ingredient molecules for drug formulation and transport systems. In this context, the focus of this review is the emergent role of surface-active ionic liquids (SAILs) in drug development and delivery.

The Impact of [C16Pyr][Amp] on the Aggressiveness in Breast and Prostate Cancer Cell Lines.

Breast (BrCa) and prostate (PCa) cancers are the most common malignancies in women and men, respectively. The available therapeutic options for these tumors are still not curative and have severe side effects. Therefore, there is an urgent need for more effective antineoplastic agents. Herein, BrCa, PCa, and benign cell lines were treated with two ionic liquids and two quinoxalines and functional experiments were performed-namely cell viability, apoptosis, cytotoxicity, and colony formation assays. At the molecular level, an array of gene expressions encompassing several molecular pathways were used to explore the impact of treatment on gene expression. Although both quinoxalines and the ionic liquid [C2OHMIM][Amp] did not show any effect on the BrCa and PCa cell lines, [C16Pyr][Amp] significantly decreased cell viability and colony formation ability, while it increased the apoptosis levels of all cell lines. Importantly, [C16Pyr][Amp] was found to be more selective for cancer cells and less toxic than cisplatin. At the molecular level, this ionic liquid was also associated with reduced expression levels of CPT2, LDHA, MCM2, and SKP2, in both BrCa and PCa cell lines. Hence, [C16Pyr][Amp] was shown to be a promising anticancer therapeutic agent for BrCa and PCa cell lines.

Recycling Old Antibiotics with Ionic Liquids.

Antibiotics are considered one of the great "miracles" of the 20th century. Now in the 21st century in the post-antibiotic era, the miracle is turning into a nightmare, due to the growing problem of the resistance of microorganisms to classic antimicrobials and the non-investment by the pharmaceutical industry in new antimicrobial agents. Unfortunately, the current COVID-19 pandemic has demonstrated the global risks associated with uncontrolled infections and the various forms of impact that such a pandemic may have on the economy and on social habits besides the associated morbidity and mortality. Therefore, there is an urgent need to recycle classic antibiotics, as is the case in the use of ionic liquids (ILs) based on antibiotics. Thus, the aim of the present review is to summarize the data on ILs, mainly those with antimicrobial action and especially against resistant strains. The main conclusions of this article are that ILs are flexible due to their ability to modulate cations and anions as a salt, making it possible to combine the properties of both and multiplying the activity of separate cations and anions. Also, these compounds have low cost methods of production, which makes it highly attractive to explore them, especially as antimicrobial agents and against resistant strains. ILs may further be combined with other therapeutic strategies, such as phage or lysine therapy, enhancing the therapeutic arsenal needed to fight this worldwide problem of antibacterial resistance. Thus, the use of ILs as antibiotics by themselves or together with phage therapy and lysine therapy are promising alternatives against pathogenic microorganisms, and may have the possibility to be used in new ways in order to restrain uncontrolled infections.

Synthesis and Antibacterial Activity of Ionic Liquids and Organic Salts Based on Penicillin G and Amoxicillin hydrolysate Derivatives against Resistant Bacteria.

The preparation and characterization of ionic liquids and organic salts (OSILs) that contain anionic penicillin G [secoPen] and amoxicillin [seco-Amx] hydrolysate derivatives and their in vitro antibacterial activity against sensitive and resistant Escherichia coli and Staphylococcus aureus strains is reported. Eleven hydrolyzed ß-lactam-OSILs were obtained after precipitation in moderate-to-high yields via the neutralization of the basic ammonia buffer of antibiotics with different cation hydroxide salts. The obtained minimum inhibitory concentration (MIC) data of the prepared compounds showed a relative decrease of the inhibitory concentrations (RDIC) in the order of 100 in the case of [C2OHMIM][seco-Pen] against sensitive S. aureus ATCC25923 and, most strikingly, higher than 1000 with [C16Pyr][seco-Amx] against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. These outstanding in vitro results showcase that a straightforward transformation of standard antibiotics into hydrolyzed organic salts can dramatically change the pharmaceutical activity of a drug, including giving rise to potent formulations of antibiotics against deadly bacteria strains.

Oxidative Stress Modulation and Radiosensitizing Effect of Quinoxaline-1,4-Dioxides Derivatives.

BACKGROUND: Quinoxaline-1,4-dioxide (QNX) derivatives are synthetic heterocyclic compounds with multiple biological and pharmacological effects. OBJECTIVE: In this study, we investigated the oxidative status of quinoxaline-1,4-dioxides derivatives in modulating melanoma and glioma cell lines, based on previous results from the research group and their capability to promote cell damage by the production of Reactive Oxygen Species (ROS). METHODS: Using in vitro cell cultures, the influence of 2-amino-3-cyanoquinoxaline-1,4-dioxide (2A3CQNX), 3- methyl-2-quinoxalinecarboxamide-1,4-dioxide (3M2QNXC) and 2-hydroxyphenazine-1,4-dioxide (2HF) was evaluated in metabolic activity, catalase activity, glutathione and 3-nitrotyrosine (3-NT) quantitation by HPLC in malignant melanocytes (B16-F10, MeWo) and brain tumor cells (GL-261 and BC3H1) submitted to radiotherapy treatments (total dose of 6 Gy). RESULTS: 2HF increased the levels of 3-NT in non-irradiated MeWo and glioma cell lines and decreased cell viability in these cell lines with and without irradiation. CONCLUSION: Quinoxaline-1,4-dioxides derivatives modulate the oxidative status in malignant melanocytes and brain tumor cell lines and exhibited a potential radiosensitizer in vitro action on the tested radioresistant cell lines.

Cinnamic Acid Conjugates in the Rescuing and Repurposing of Classical Antimalarial Drugs.

Cinnamic acids are compounds of natural origin that can be found in many different parts of a wide panoply of plants, where they play the most diverse biological roles, often in a conjugated form. For a long time, this has been driving Medicinal Chemists towards the investigation of the therapeutic potential of natural, semi-synthetic, or fully synthetic cinnamic acid conjugates. These efforts have been steadily disclosing promising drug leads, but a wide chemical space remains that deserves to be further explored. Amongst different reported approaches, the combination or conjugation of cinnamic acids with known drugs has been addressed in an attempt to produce either synergistic or multi-target action. In this connection, the present review will focus on efforts of the past decade regarding conjugation with cinnamic acids as a tool for the rescuing or the repurposing of classical antimalarial drugs, and also on future perspectives in this particular field of research.

A Novel Approach for Bisphosphonates: Ionic Liquids and Organic Salts from Zoledronic Acid.

Novel ionic liquids and organic salts based on mono- or dianionic zoledronate and protonated superbases, choline and n-alkylmethylimidazolium cations, were prepared and characterized by spectroscopic and thermal analyses. Most of the prepared salts display amorphous structures and very high solubility in water and saline solutions, especially the dianionic salts. Among the zoledronate-based ionic compounds, those containing choline [Ch] and methoxyethylmethylimidazolium [C3 OMIM] cations appear to have significant cytotoxicity against human osteosarcoma cells (MG63) and low toxicity toward healthy skin fibroblast cells. Because osteosarcoma is a bone pathology characterized by an increase in bone turnover rate, the results presented herein may be a promising starting point for the development of new ionic pharmaceutical drugs against osteosarcoma.

ESBL and AmpC ß-Lactamases in Clinical Strains of Escherichia coli from Serra da Estrela, Portugal.

Background and Objectives: Given the considerable spatial, temporal, and ecological factors, heterogeneity, which affects emergency response, persistence, and dissemination of genetic determinants that confer microorganisms their resistance to antibiotics, several authors claim that antibiotics' resistance must be perceived as an ecological problem. The aim of this study was to determine the prevalence of broad-spectrum bla genes, not only Extended-spectrum ß-lactamases (ESBL) but also AmpC-types, in clinical strains of Escherichia coli isolated from Portugal (in the highest region of the country, Serra da Estrela) to disclose susceptibility profiles among different genotypes, and to compare the distribution of bla genes expressing broad-spectrum enzymes. Materials and Methods: Clinical strains of Escherichia coli presenting resistance to third generation (3G) cephalosporins and susceptibility to inhibition by clavulanic acid were studied by means of phenotypic and molecular profiling techniques for encoding ß-lactamases genes. Results: Strains were mainly isolated from hospital populations (97%). Molecular analysis enabled the detection of 49 bla genes, in which 55% (27/49) were identified as blaOXA-1-like, 33% (16/49) as blaCTX-M-group-1, 10% (5/49) as blaTEM, and 2% (1/49) were identified as genes blaCIT (AmpC). Among all blaOXA-1-like detected, about 59% of strains expressed at least another bla gene. Co-production of ß-lactamases was observed in 40% of strains, with the co-production of CTX-M group 1 and OXA-1-like occurring as the most frequent. Conclusions: This is the first study using microorganisms isolated from native people from the highest Portuguese mountain regions, showing an unprecedent high prevalence of genes blaOXA-1-like in this country.

Quinoxaline-1,4-dioxide derivatives inhibitory action in melanoma and brain tumor cells.

Aim: Quinoxaline-1,4-dioxide derivatives are synthetic heterocyclic compounds with multiple biological and pharmacological effects. In this study, we investigated the bioactivity of five quinoxaline-1,4-di-N-oxides derivatives in different animal cell lines. Materials & methods: Using in vitro cell cultures, we evaluated the influence of quinoxaline-1,4-dioxide, 2-methylquinoxaline-1,4-dioxide, 2-amino-3-cyanoquinoxaline-1,4-dioxide, 3-methyl-2-quinoxalinecarboxamide-1,4-dioxide and 2-hydroxyphenazine-N,N-dioxide (2HF) in the viability, migration and proliferation of nonmalignant (3T3-L1 and human dermal microvascular endothelial cell) and malignant (B16-F10, MeWo, GL-261 and BC3H1) cell lines. Results: The viability IC50 concentrations for each quinoxaline-1,4-di-N-oxide derivative were calculated, and a concomitant reduction of migration and proliferation was observed mainly in malignant cell lines. Conclusion: 2HF exhibited potent anti-viability, anti-migration and anti-proliferative actions selectively in tumor cells, nevertheless more studies are required to further investigate 2HF promising biologic effects.

Differential effects of antiepileptic drugs on human bone cells.

Antiepileptic drugs (AED) have been associated to in vivo deleterious consequences in bone tissue. The present work aimed to characterize the cellular and molecular effects of five different AED on human osteoclastogenesis and osteblastogenesis. It was observed that the different drugs had the ability to differentially modulate both processes, in a way dependent on the identity and dose of the AED. Shortly, valproic acid stimulated either osteoclastogenesis and osteoblastogenesis, whereas carbamazepine, gabapentin, and lamotrigine revealed an opposite behavior; topiramate elicited a decrease of osteoclast development and an increase in osteoblast differentiation. This is the first report describing the direct effects of different AED on human primary bone cells, which is a very important issue, because these drugs are usually consumed in long-term therapeutics, with acknowledged in vivo effects in bone tissue.

Prevalence of Antibiotic Resistance Genes in Multidrug-Resistant Enterobacteriaceae on Portuguese Livestock Manure.

The exposure of both crop fields and humans to antibiotic-resistant bacteria in animal excreta is an emergent concern of the One Health initiative. This study assessed the contamination of livestock manure from poultry, pig, dairy farms and slaughterhouses in Portugal with resistance determinants. The resistance profiles of 331 Enterobacteriaceae isolates to eight ß-lactam (amoxicillin, cefoxitin, cefotaxime, cefpirome, aztreonam, ceftazidime, imipenem and meropenem) and to five non-ß-lactam antibiotics (tetracycline (TET), trimethoprim/sulfamethoxazole (SXT), ciprofloxacin (CIP), chloramphenicol (CHL) and gentamicin) was investigated. Forty-nine integron and non-ß-lactam resistance genes were also screened for. Rates of resistance to the 13 antibiotics ranged from 80.8% to 0.6%. Multidrug resistance (MDR) rates were highest in pig farm samples (79%). Thirty different integron and resistance genes were identified. These were mainly associated with resistance to CHL (catI and catII), CIP (mainly, qnrS, qnrB and oqx), TET (mainly tet(A) and tet(M)) and SXT (mostly dfrIa group and sul3). In MDR isolates, integron presence and non-ß-lactam resistance to TET, SXT and CHL were positively correlated. Overall, a high prevalence of MDR Enterobacteriaceae was found in livestock manure. The high gene diversity for antibiotic resistance identified in this study highlights the risk of MDR spread within the environment through manure use.

Chemistry, bioactivities, extraction and analysis of azadirachtin: State-of-the-art.

Azadirachta indica A. Juss. (Neem) is an Indian tree recognized for its activity as pesticide, as well as several pharmacological properties. Among the various compounds already isolated and studied from Neem tree, azadirachtin (AZA) was identified as the main bioactive compound. Azadirachtin can be found at different parts of the Neem plant but assumes its maximum concentration at the seed level. This compound features a quite complex chemical structure, which justifies the 20-plus-year difficulty to identify the synthetic pathway that subsequently permitted to carry out its artificial synthesis. Azadirachtin is widely used as a basis for production of biopesticides; nevertheless, other properties have been recognized for this substance, among which the anticancer and antimalarial activity stand out. The methods available for azadirachtin extraction are diverse, including solid-liquid extraction and extraction with solvents at high or low temperatures. Alcohol based solvents are associated with higher extraction yields and are therefore preferred for the isolation of azadirachtin from plant parts. Clean-up of the extracts is generally required for further purification. The highest azadirachtin levels have been obtained from Neem seeds but concentration values present a large variation between batches. Therefore, in addition to extraction procedures, it is essential to establish routine methods for azadirachtin identification and quantification. Chromatography-based techniques are preferably selected for detection and quantification of azadirachtin in plant matrices. Overall, this process will guarantee a future reproducible, safe and effective use of the extracts in formulations for commercial applications.

Ionic Liquids for Topical Delivery in Cancer.

The unique properties of ionic liquids make them quite appealing for diverse applications, from "green" solvents (1st generation ionic liquids) to finely tuned materials (2nd generation ionic liquids). A decade ago, a 3rd generation of ionic liquids emerged which is focused on their prospective clinical applications, either as drugs per se or as adjuvants in drug formulations. In recent years, research focused on the use of ionic liquids for topical drug delivery has been increasing and holds great promise towards clinical application against skin cancers. This article highlights the growing relevance of ionic liquids in medicinal chemistry and pharmaceutical technology, which is opening new windows of opportunity.