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1.
Turkiye Parazitol Derg ; 46(2): 97-101, 2022 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-35604185

RESUMO

Objective: Leishmaniasis is the second deadliest parasitic disease in the World Health Organisation's list of neglected diseases, following malaria. Cutaneous leishmaniasis (CL) is the most common form of the disease and it is one of the few communicable diseases with increasing incidence rates owing to factors like armed conflicts and climate change. CL can be divided into two major groups: Acute CL (ACL) and chronic CL (CCL). The aim of this study was to compare the in vitro efficacy of miltefosine and pentavalent antimony compounds in the CCL patient samples. Methods: Five isolates previously isolated from 5 CCL patients were included in this study. Genotyping is performed using internal transcribed spacer 1 (ITS 1) gene region real-time PCR. In vitro drug efficacy tests were applied to determine their activity against meglumine antimoniate (MA) and miltefosine. Serial dilutions (512, 256, 128, 64, 32, 16, 8 and 4 µg/mL) prepared from MA and miltefosine were prepared in 96-well flat-bottom cell culture plates and incubated at 24 °C for 48 hours. The efficacy of the drug on Leishmania spp. promastigotes after 24 and 48 hours was evaluated by hemocytometer slide and XTT cell viability test. Results: All of the samples were genotyped as L. tropica. Evaluation of 24 and 48 hours showed, 128 µg/mL and 256 µg/mL and 32 µg/mL and 64 µg/mL concentrations of miltefosine and MA were enough to kill all the promastigotes respectively. The results of the hemocytometer slide and XTT were consistent. Conclusion: There are no studies investigating the in vitro efficacy of miltefosine with the CCL patient group. To overcome the treatment challenges experienced in this special patient group, more studies are needed. According to our results, it is concluded that miltefosine is efficient for the treatment of CCL and further clinical studies with miltefosine will reveal valuable data.


Assuntos
Antiprotozoários , Leishmaniose Cutânea , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Humanos , Leishmaniose Cutânea/tratamento farmacológico , Antimoniato de Meglumina/farmacologia , Antimoniato de Meglumina/uso terapêutico , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico
2.
Biomater Sci ; 10(10): 2665-2672, 2022 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-35420601

RESUMO

Radiation therapy is a powerful approach for cancer treatment due to its low invasiveness. The development of radiation sensitizers is of great importance as they assist in providing radiation therapy at a low dose. In this study, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-modified gold nanocomposites of different shapes were created using the grafting-to approach to serve as a novel radiation sensitizer with high cellular uptake. The effect of the shape of the nanocomposite on cellular uptake by the breast cancer cell line MCF-7 was also investigated. The PMPC-modified gold nanostars showed the highest cellular uptake compared to the other gold nanocomposites (spheres and rods), whereas cell cytotoxicity was negligible among all candidates. Furthermore, the therapeutic effect of radiation of PMPC-modified nanostars was the highest among all the gold nanocomposites. These results clearly indicate that the shape of the gold nanocomposite is an important parameter for cellular uptake and radiation sensitizing effects in breast cancer cells.


Assuntos
Neoplasias da Mama , Nanocompostos , Radiossensibilizantes , Neoplasias da Mama/radioterapia , Feminino , Ouro , Humanos , Fosforilcolina/farmacologia , Polímeros , Ácidos Polimetacrílicos
3.
ACS Appl Mater Interfaces ; 14(7): 9557-9569, 2022 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-35144379

RESUMO

The rapid accumulation of dead bacteria or protein on a bactericidal surface can reduce the effectiveness of the modified surface and alter its biocidal activity by shielding the surface biocide functional groups, promoting microbial attachment and subsequent biofilm formation. Thus, the alteration of biocidal activity due to biofilm formation can cause serious trouble including severe infection or implant or medical device failure leading to death. Therefore, developing a smart self-cleaning surface is of great interest. Ideally, such a surface can not only kill the attached microbials but also release the dead cells and foulants from the surface under a particular incitement on demand. In this project, a sugar-responsive self-cleaning coating has been developed by forming covalent boronic ester bonds between catechol groups from polydopamine and a benzoxaborole pendant from zwitterionic and cationic polymers. To incorporate antifouling properties and enhance the biocompatibility of the coating, bioinspired zwitterionic compound 2-methacryloyloxyethyl phosphorylcholine (MPC) was chosen and benzoxaborole pendant containing zwitterionic polymer poly(MPC-st-MAABO) (MAABO: 5-methacrylamido-1,2-benzoxaborole) was synthesized. Additionally to impart antibacterial properties to the surface, a quaternary ammonium containing cationic polymer poly(2-(methacryloyloxy)ethyl trimethylammonium (META)-st-MAABO)) was synthesized. These synthesized polymers were covalently grafted to a polydopamine (PDA) coated surface by forming a strong cyclic boronic ester complex with a catechol group of the PDA layer endowing the surface with bacteria contact-killing properties and capturing specific protein. After the addition of cis-diol containing competitive molecules, i.e., saccharides/sugars, this boronic ester complex with a catechol group of PDA was replaced and the attached polymer layer was cleaved from the surface, resulting in the release of both absorbed protein and live/killed bacteria electrostatically attached to the polymer layer. This dynamic self-cleaning surface can be a promising material for biomedical applications avoiding the gathering of dead cells and debris that are typically encountered on a traditional biocidal surface.


Assuntos
Incrustação Biológica , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias , Incrustação Biológica/prevenção & controle , Dopamina/farmacologia , Fosforilcolina/química , Fosforilcolina/farmacologia , Propriedades de Superfície
4.
Biochim Biophys Acta Biomembr ; 1864(5): 183872, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35085568

RESUMO

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to characterize the components of the Mycobacterium abscessus massiliense cell envelope and their interactions with amphotericin B (AmB), miltefosine (MIL), and nerolidol (NER). Spin labels analogous to stearic acid and phosphatidylcholine (PC) were distributed on an envelope layer with fluidity comparable to other biological membranes, probably the mycobacterial cell wall, because after treatment with AmB a highly rigid spectral component was evident in the EPR spectra. Methyl stearate analogue spin labels found a much more fluid membrane and did not detect the presence of AmB, except for at very high drug concentrations. Unlike other spin-labeled PCs, the TEMPO-PC spin probe, with the nitroxide moiety attached to the choline of the PC headgroup, also did not detect the presence of AmB. On the other hand, the steroid spin labels were not distributed across the membranes of M. abscessus and, instead, were concentrated in some other location of the cell envelope. Both MIL and NER compounds at 10 µM caused increased fluidity in the cell wall and plasma membrane. Furthermore, NER was shown to have a remarkable ability to extract lipids from the mycobacterial cell wall. The EPR results suggest that the resistance of mycobacteria to the action of AmB must be related to the fact that this drug does not reach the bacterial plasma membrane.


Assuntos
Anfotericina B/farmacologia , Antibacterianos/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Mycobacterium abscessus/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Sesquiterpenos/farmacologia , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Parede Celular/química , Parede Celular/efeitos dos fármacos , Óxidos N-Cíclicos/química , Testes de Sensibilidade Microbiana , Mycobacterium abscessus/química , Mycobacterium abscessus/metabolismo , Fosfatidilcolinas/química , Fosforilcolina/farmacologia , Marcadores de Spin , Ácidos Esteáricos/química
5.
Int J Antimicrob Agents ; 59(1): 106459, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34695563

RESUMO

Miltefosine is an alkylphosphocholine agent with a broad spectrum of antiparasitic properties. For over two decades, miltefosine has remained the only oral drug licensed and used in the treatment of the neglected tropical disease, leishmaniasis. The last extensive review of the pharmacology of miltefosine was published in 2012. Additional data on the clinical pharmacokinetics (PK) and pharmacodynamics (PD) of miltefosine have become available in the last decade, and there are ongoing and future studies in this area. Miltefosine PK are characterized by slow absorption and elimination, resulting in accumulation of drug in plasma until the end of treatment. Several recent studies established exposure-response relationships for various regimens of miltefosine in the treatment of visceral and cutaneous leishmaniasis, leading to the identification of PK parameters predictive of clinical relapse and outcome. This review provides an update on the most recent developments in the area of clinical pharmacology of miltefosine, including a discussion of the current dosing regimens.


Assuntos
Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Leishmaniose Cutânea/tratamento farmacológico , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Humanos
6.
Drug Deliv Transl Res ; 12(1): 180-196, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-33502733

RESUMO

Cutaneous leishmaniasis (CL) is a neglected tropical disease endemic in ~ 90 countries, with an increasing incidence. Presently available pharmacotherapy implies the systemic administration of moderately/very toxic drugs. Miltefosine (Milt) is the only FDA-approved drug to treat CL via the oral route (Impavido®). It produces side effects; in particular, teratogenic effects are of concern. A topical treatment would have the great advantage of minimising the systemic circulation of the drug, preventing side effects. We prepared dispersions containing Milt and liposomes of different compositions to enhance/modulate trans-epidermal penetration and evaluated in vitro and in vivo efficacy and toxicity, in vitro release rate of the drug and particles size stability with time. Treatments were topically administered to BALB/c mice infected with Leishmania (Leishmania) amazonensis. The dispersions containing 0.5% Milt eliminated 99% of the parasites and cured the lesions with a complete re-epithelisation, no visible scar and re-growth of hair. Fluid liposomes decreased the time to heal the lesion and the time needed to eliminate viable amastigotes from the lesion site. Relapse of the infection was not found 1 month after treatment in any case. Ultraflexible liposomes on the other hand had no significant in vitro effect but decreased in vivo efficacy. A topical Milt formulation including fluid liposomes seems a promising treatment against CL.


Assuntos
Leishmania , Leishmaniose Cutânea , Animais , Leishmaniose Cutânea/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Modelos Teóricos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico
7.
Parasit Vectors ; 14(1): 599, 2021 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-34886876

RESUMO

BACKGROUND: Leishmania infantum is the most important etiological agent of visceral leishmaniasis in the Americas and Mediterranean region, and the dog is the main host. Miltefosine was authorized to treat canine leishmaniasis (CanL) in Brazil in 2017, but there is a persistent fear of the emergence of parasites resistant not only to this drug but, through cross-resistance mechanisms, also to meglumine antimoniate and amphotericin B. Additionally, the literature shows that acquisition of resistance is followed by increased parasite fitness, with higher rates of proliferation, infectivity and metacyclogenesis, which are drivers of parasite virulence. In this context, the aim of this study was to analyze the impact of treating a dog with miltefosine and allopurinol on the generation of parasites resistant to miltefosine, amphotericin B and meglumine antimoniate. METHODS: In vitro susceptibility tests were conducted against miltefosine, amphotericin B and meglumine antimoniate with T0 (parasites isolated from a dog before treatment with miltefosine plus allopurinol), T1 (after 1 course of treatment) and T2 (after 2 courses of treatment) isolates. The rates of cell proliferation, infectivity and metacyclogenesis of the isolates were also evaluated. RESULTS: The results indicate a gradual increase in parasite resistance to miltefosine and amphotericin B with increasing the number of treatment courses. An increasing trend in the metacyclogenesis rate of the parasites was also observed as drug resistance increased. CONCLUSION: The data indicates an increased L. infantum resistance to miltefosine and amphotericin B after the treatment of a dog with miltefosine plus allopurinol. Further studies with a larger number of L. infantum strains isolated from dogs with varied immune response profiles and undergoing different treatment regimes, are advocated.


Assuntos
Anfotericina B/farmacologia , Antiprotozoários/farmacologia , Doenças do Cão/parasitologia , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Fosforilcolina/análogos & derivados , Alopurinol/uso terapêutico , Animais , Antiprotozoários/uso terapêutico , Doenças do Cão/tratamento farmacológico , Cães , Resistência a Medicamentos , Feminino , Leishmaniose Visceral/tratamento farmacológico , Antimoniato de Meglumina/uso terapêutico , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico
8.
Clin Transl Med ; 11(11): e552, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34841679

RESUMO

BACKGROUND: Lipid rafts (LRs), cholesterol-enriched microdomains on cell membranes, are increasingly viewed as signalling platforms governing critical facets of cancer progression. The phenotype of cancer stem-like cells (CSCs) presents significant hurdles for successful cancer treatment, and the expression of several CSC markers is associated with LR integrity. However, LR implications in CSCs remain unclear. METHODS: This study evaluated the biological and molecular functions of LRs in colorectal cancer (CRC) by using an LR-disrupting alkylphospholipid (APL) drug, miltefosine. The mechanistic role of miltefosine in CSC inhibition was examined through normal or tumour intestinal mouse organoid, human CRC cell, CRC xenograft and miltefosine treatment gene expression profile analyses. RESULTS: Miltefosine suppresses CSC populations and their self-renewal activities in CRC cells, a CSC-targeting effect leading to irreversible disruption of tumour-initiating potential in vivo. Mechanistically, miltefosine reduced the expression of a set of genes, leading to stem cell death. Among them, miltefosine transcriptionally inhibited checkpoint kinase 1 (CHEK1), indicating that LR integrity is essential for CHEK1 expression regulation. In isolated CD44high CSCs, we found that CSCs exhibited stronger therapy resistance than non-CSC counterparts by preventing cell death through CHEK1-mediated cell cycle checkpoints. However, inhibition of the LR/CHEK1 axis by miltefosine released cell cycle checkpoints, forcing CSCs to enter inappropriate mitosis with accumulated DNA damage and resulting in catastrophic cell death. CONCLUSION: Our findings underscore the therapeutic potential of LR-targeting APLs for CRC treatment that overcomes the therapy-resistant phenotype of CSCs, highlighting the importance of the LR/CHEK1 axis as a novel mechanism of APLs.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Microdomínios da Membrana/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/fisiopatologia , Modelos Animais de Doenças , Camundongos , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico
9.
Biol Pharm Bull ; 44(11): 1717-1723, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34719648

RESUMO

Acetylcholine (ACh), a quaternary ammonium cation, is known as one of the itch inducer in atopic dermatitis (AD), an inflammatory skin disease with intense itching. Previous research has reported accumulation of ACh in lesional site of AD patients. Generally, ACh is metabolized by cholinesterase (ChE). Therefore, one of the causes of ACh accumulation may be the suppression of ChE activity. Increased levels of the multifunctional bioactive sphingolipid sphingosylphosphorylcholine (SPC) have also been detected in AD. Since SPC possesses a quaternary ammonium cation, like ACh, it is possible that SPC affects the activity of ChE catalyzing ACh metabolization. We investigated whether SPC influences the activity of ChE by performing enzymatic analysis of ChE in the presence of SPC. We found that SPC strongly suppressed acetylcholinesterase (AChE) activity, but the suppression of butyrylcholinesterase by SPC was quite low. The Michaelis constant (Km) of AChE in the presence of SPC increased, and the maximum velocity (Vmax) decreased, indicating that SPC acts as mixed-type inhibitor for AChE. The analysis of SPC analogs clarified the importance of both the quaternary ammonium cation and the carbon chain length of SPC for the AChE inhibitory effect and showed that SPC was unique in AChE inhibition among the sphingolipids in this study. These findings indicate a novel function of SPC on AChE inhibition. Thus, the inhibition activity of SPC may be a factor in the increase of ACh in AD.


Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Fosforilcolina/análogos & derivados , Esfingosina/análogos & derivados , Relação Dose-Resposta a Droga , Humanos , Neostigmina/farmacologia , Fosforilcolina/farmacologia , Rivastigmina/farmacologia , Esfingosina/farmacologia
10.
Int J Mol Sci ; 22(21)2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34769032

RESUMO

BACKGROUND: Fluoroquinolones (FQs) are potent antimicrobials with multiple effects on host cells and tissues. Although FQs can attenuate cancer invasion and metastasis, the underlying molecular mechanisms remain unclear. Matrix metalloproteinase-9 (MMP-9) has functional roles in tumor angiogenesis, invasion, and metastasis, and is associated with cancer progression and poor prognosis, suggesting that inhibitors of MMP-9 activity and transcription are prime candidates for cancer therapy. Despite numerous preclinical data supporting the use of MMP-9 inhibitors as anticancer drugs, the few available examples are not therapeutically useful due to low specificity and off-target effects. We examined the effects of FQs on MMP-9 production in cancer cells following transforming growth factor beta (TGF-ß) and phorbol 12-myristate 13-acetate (PMA) stimulation. EXPERIMENTAL APPROACHES: Using confluent cultures of HepG2 and A549 cells, the effects of FQs (ciprofloxacin, levofloxacin, clinafloxacin, gatifloxacin, and enrofloxacin) on TGF-ß and PMA-induced MMP-9 mRNA expression and production were studied in RNA extracts and culture supernatants, respectively. FQs specifically abrogated TGF-ß and PMA-induced MMP-9 levels and activity in a concentration and time-dependent manner, without affecting other MMPs or proteins involved in epithelial-mesenchymal transition. Additionally, FQs inhibited TGF-ß and PMA-induced cell migration via p38 and cyclic AMP signaling pathways. CONCLUSIONS AND IMPLICATIONS: Overall, we demonstrated that FQs inhibit cancer cell migration and invasion by downregulating MMP-9 expression and revealed the cellular mechanisms underlying their potential value in cancer treatment.


Assuntos
Antibacterianos/farmacologia , Fluoroquinolonas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Metaloproteinase 9 da Matriz/metabolismo , Fosforilcolina/análogos & derivados , Ácidos Polimetacrílicos/farmacologia , Quinolonas/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Células A549 , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Reposicionamento de Medicamentos/métodos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Pulmonares/metabolismo , Invasividade Neoplásica/patologia , Fosforilcolina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Bioengineered ; 12(2): 10264-10284, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34720043

RESUMO

Chronic skin ulcers are a primary global health problem. Velvet antler polypeptide (VAP) regulates endothelial cell migration and angiogenic sprout. Adipose-derived stem cells (ADSCs) are reported to make pivotal impacts upon wound healing. This study aimed to explore the role of VAP combined with ADSCs in wound healing of chronic skin ulcers. The effect of VAP on phenotypes of ADSCs, and VAP (PLGA microspheres) combining with ADSCs on wound healing of chronic skin ulcers in vivo was evaluated. VAP generally promoted the proliferation, migration and invasion of ADSCs, and ADSC-induced angiogenesis in human umbilical vein endothelial cells (HUVECs) through PI3K/Akt/HIF-1α pathway. VAP-PLGA (PLGA microspheres) enhanced the promoting effect of ADSCs on wound healing, pathological changes, and angiogenesis in chronic skin ulcers in vivo. VAP-PLGA intensified the effect of ADSCs on up-regulating the levels of p-PI3K/PI3K, p-Akt/Akt, HIF-1α, vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), C-X-C motif chemokine receptor 4 (CXCR4), angiopoietin-4 (Ang-4), VEGF receptor (VEGFR), and transforming growth factor-ß1 (TGF-ß1), and down-regulating the levels of interleukin-1 ß (IL-1ß), IL-18 and IL-6 in wound tissues in chronic skin ulcers in vivo. Collectively, VAP promoted the growth, migration, invasion, and angiogenesis of ADSCs through activating PI3K/Akt/HIF-1α pathway, and VAP-PLGA enhanced the function of ADSCs in promoting wound healing in vivo, which was associated with angiogenesis, inflammation inhibition, and dermal collagen synthesis.


Assuntos
Tecido Adiposo/citologia , Microesferas , Peptídeos/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Transdução de Sinais , Úlcera Cutânea/terapia , Células-Tronco/citologia , Cicatrização , Animais , Chifres de Veado , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Cromonas/farmacologia , Doença Crônica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Morfolinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo
12.
Future Microbiol ; 16: 1153-1160, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34615383

RESUMO

Aim: To evaluate the activity of miltefosine (MFS), in its free form or loaded-alginate nanoparticles (MFS-AN), alone or combined with voriconazole (VRC) on Aspergillus fumigatus and Aspergillus flavus. Materials & methods: A broth microdilution assay was used for the susceptibility testing of Aspergillus isolates, and the antifungal efficacy was assessed using the aspergillosis model in Galleria mellonella larvae. Results: The in vitro synergistic effect of MFS with VRC was observed only against A. fumigatus, whereas both combined therapies (MFS + VRC and MFS-AN + VRC) showed synergism in reducing the larval mortality rate and fungal burden in the larvae infected by A. fumigatus and A. flavus. Conclusions: MFS and MFS-AN combined with VRC may be an important strategy for improving antifungal therapy against aspergillosis.


Assuntos
Antifúngicos , Aspergillus flavus/efeitos dos fármacos , Aspergillus fumigatus/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Voriconazol , Alginatos , Antifúngicos/farmacologia , Portadores de Fármacos , Sinergismo Farmacológico , Nanopartículas , Fosforilcolina/farmacologia , Voriconazol/farmacologia
13.
Mater Sci Eng C Mater Biol Appl ; 129: 112367, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34579886

RESUMO

We developed a hemocompatible, bio-inspired, multivalent, polymeric-chelating assembly based on the poly(2-methacryloyloxyethyl phosphorylcholine)-b-poly(serinyl acrylate) (PMPC-b-PserA) zwitterionic diblock copolymer. Functional PMPC-b-PserA was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization to catch and encapsulate free copper ions (Cu2+) in a solution. PMPC with an identical polar group to phospholipids exhibits high hydrophilicity and fouling resistance against non-specific adsorption, and inertness to the metal ions. On the other hand, PserA with pendant groups of amino acids possesses a strong capability to react with Cu2+ by coordination interaction. Therefore, when PMPC-b-PserA was brought into contact with Cu2+, a hydrophobic core with multiple coordination "bridges" between polymers and Cu2+ was formed, leading to self-assembly of core-shell polymer-metal nanoparticles. As a result, free Cu2+ ions can be removed from the solution to prevent damage to cells and tissues. The synthesis and chemical structure of PMPC-b-PserA were characterized, and the formation of self-assembled polymer-Cu2+ nanoparticles and colloidal stability were analyzed. More importantly, the detoxification of PMPC-b-PserA in presence of Cu2+ with fibroblast cells was demonstrated by increased cell viability >80%. In addition, the hemolysis, which occurred due to disruption of RBC membranes by free Cu2+, was effectively suppressed by adding PMPC-b-PserA. The bio-inspired and biocompatible chelating agent of PMPC-b-PserA provides a new treatment approach to encapsulate and detoxify heavy metals in complex media for chelation therapy.


Assuntos
Cobre , Hemólise , Quelantes/farmacologia , Humanos , Metacrilatos , Micelas , Fosforilcolina/farmacologia , Polímeros , Ácidos Polimetacrílicos
14.
Naunyn Schmiedebergs Arch Pharmacol ; 394(11): 2223-2232, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34406420

RESUMO

The molecular chaperone HSP90 facilitates the maturation of newly synthesized and unfolded proteins. The client proteins of HSP90 are involved in many processes of cancer occurrence and development, and therefore, HSP90 is considered as a promising target for the development of anticancer drugs. In contrast to N-terminal inhibitor, C-terminal inhibitor does not induce the pro-survival heat shock response. In order to get novel HSP90 C-terminal inhibitors and more evidences that HSP90 inhibitors could be applied in the therapy of cancer, we conducted a virtual screening toward HSP90 C-terminus from FDA-approved drugs. In this study, miltefosine and octenidine were identified as new HSP90 inhibitors. Miltefosine and octenidine exhibited strong and broad-spectrum anticancer activity and inhibited the proliferation of cancer cell by promoting apoptosis. Western blotting analysis revealed that miltefosine and octenidine significantly down-regulated the expression levels of HSP90 client proteins including p-AKT, CDK6, and ERK, and did not induce overexpression of heat shock proteins including HSP70 and HSP90 in MCF-7 cells. These results were in accordance with the characteristics of HSP90 C-terminal inhibitor. In conclusion, miltefosine and octenidine could disrupt the molecular chaperone function of HSP90, and thus, their strong and broad-spectrum anticancer activity is at least in part attributed to the inhibition activity against HSP90.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Iminas/farmacologia , Fosforilcolina/análogos & derivados , Piridinas/farmacologia , Células A549 , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Células MCF-7 , Neoplasias/tratamento farmacológico , Fosforilcolina/farmacologia
15.
Tissue Eng Regen Med ; 18(6): 953-962, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34460064

RESUMO

BACKGROUND: One crucial factor in skin tissue engineering is to understand the hydration and barrier property of skin. We investigated the skin hydration and stabilization strategy of inter-lamellar structure of stratum corneum (SC) using poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC). METHODS: The unique hydration and stabilization potency of PMPC on the barrier function of the SC examined using freshly excised hairless mouse skin as a model membrane and the relationship between the stabilization of the lipid lamellar bilayer (LLB) and its enhanced water holding capacity was established. RESULTS: Differential scanning calorimeter based on the phase-transition temperature of lipid domain of SC demonstrated that PMPC stabilized the LLB. The ratio of the heat of lipid phase transition (△H) of SC exposed to water and PMPC for 24 h was 1.51. X-ray crystallography showed the presence of well- organized lipids in intercellular membranes exhibiting short and long periodicity of lamellar phases. The peak at 4.4 nm attributed to the long periodicity phase (LPP) was missing in water-treated SC, where, the presence of 4.2- 4.4 nm peak in PMPC treated SC indicated that PMPC stabilized LPP. Transmission electron microscopy study demonstrated that the LLB structure became more rigid and orderly in PMPC treated SC. CONCLUSION: The unique ion paired structure of PMPC enhances the barrier function of the SC by stabilizing LLB structure and hydration by inducing weakly bound water. The unique hydration state and stabilization effect from extended water exposure could provide a valuable information to prepare reliable artificial skin matrix and skin tissue.


Assuntos
Bicamadas Lipídicas , Fosforilcolina , Animais , Epiderme , Metacrilatos/química , Metacrilatos/farmacologia , Camundongos , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/farmacologia
16.
Mol Biol Rep ; 48(9): 6313-6321, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34390443

RESUMO

BACKGROUNDS: Epithelial mesenchymal transition (EMT) is a critical process involved in the invasion and metastasis of cancer, including lung cancer (LC). Transforming growth factor (TGF)-ß is one of factors capable of inducing EMT. Polyinosinic-polycytidylic acid (polyI:C), a synthetic agonist for toll-like receptor (TLR) 3, can enhance immune responses and has been used as an adjuvant for cancer vaccines; however, it remains unclear whether it influences other process, such as EMT. In the present study, we examined the effects of polyI:C on TGF-ß-treated A549 human LC cells. METHODS AND RESULTS: By in vitro cell proliferation assay, polyI:C showed no effect on the growth of A549 cells treated with TGF-ß1 at the concentration range up to 10 µg/ml; however, it markedly suppressed the motility in a cell scratch and a cell invasion assay. By Western blotting, polyI:C dramatically decreased TGF-ß1-induced Ak strain transforming (Akt) phosphorylation and increased phosphatase and tensin homologue (PTEN) expression without affecting the Son of mothers against decapentaplegic (Smad) 3 phosphorylation or the expression level of E-cadherin, N-cadherin or Snail, indicating that polyI:C suppressed cell motility independently of the 'cadherin switching'. The Akt inhibitor perifosine inhibited TGF-ß1-induced cell invasion, and the PTEN-specific inhibitor VO-OHpic appeared to reverse the inhibitory effect of polyI:C. CONCLUSION: PolyI:C has a novel function to suppress the motility of LC cells undergoing EMT by targeting the phosphatidylinositol 3-kinase/Akt pathway partly via PTEN and may prevent or reduce the metastasis of LC cells.


Assuntos
Adenocarcinoma Bronquioloalveolar/metabolismo , Movimento Celular/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Células A549 , Adenocarcinoma Bronquioloalveolar/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/patologia , Compostos Organometálicos/farmacologia , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Receptor 3 Toll-Like/agonistas
17.
mBio ; 12(4): e0145821, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34372704

RESUMO

Aspergillus fumigatus is an important fungal pathogen and the main etiological agent of aspergillosis, a disease characterized by a noninvasive process that can evolve to a more severe clinical manifestation, called invasive pulmonary aspergillosis (IPA), in immunocompromised patients. The antifungal arsenal to threat aspergillosis is very restricted. Azoles are the main therapeutic approach to control IPA, but the emergence of azole-resistant A. fumigatus isolates has significantly increased over recent decades. Therefore, new strategies are necessary to combat aspergillosis, and drug repurposing has emerged as an efficient and alternative approach for identifying new antifungal drugs. Here, we used a screening approach to analyze A. fumigatus in vitro susceptibility to 1,127 compounds. A. fumigatus was susceptible to 10 compounds, including miltefosine, a drug that displayed fungicidal activity against A. fumigatus. By screening an A. fumigatus transcription factor null library, we identified a single mutant, which has the smiA (sensitive to miltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and ΔsmiA strains and chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-Seq) of an SmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipid pathway that are directly or indirectly regulated by SmiA. Sphingolipid analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of SmiA represents the first genetic element described and characterized that plays a direct role in miltefosine response in fungi. IMPORTANCE The filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis, and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over recent decades. Repositioning or repurposing drugs already available on the market is an interesting and faster opportunity for the identification of novel antifungal agents. By using a repurposing strategy, we identified 10 different compounds that impact A. fumigatus survival. One of these compounds, miltefosine, demonstrated fungicidal activity against A. fumigatus. The mechanism of action of miltefosine is unknown, and, aiming to get more insights about it, we identified a transcription factor, SmiA (sensitive to miltefosine), important for miltefosine resistance. Our results suggest that miltefosine displays antifungal activity against A. fumigatus, interfering in sphingolipid biosynthesis.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Ensaios de Triagem em Larga Escala , Fosforilcolina/análogos & derivados , Bibliotecas de Moléculas Pequenas/farmacologia , Esfingolipídeos/metabolismo , Animais , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Aspergillus fumigatus/química , Aspergillus fumigatus/patogenicidade , Farmacorresistência Fúngica , Larva/efeitos dos fármacos , Larva/microbiologia , Testes de Sensibilidade Microbiana , Mariposas/efeitos dos fármacos , Mariposas/microbiologia , Fenótipo , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Virulência
18.
Biochim Biophys Acta Biomembr ; 1863(11): 183698, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34283999

RESUMO

Hexadecylphosphocholine (HePC, Miltefosine) is a drug from the class of alkylphosphocholines with an antineoplastic and antiprotozoal activity. We previously reported that HePC uptake from thermosensitive liposomes (TSL) containing 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) into cancer cells is accelerated at mild hyperthermia (HT) resulting in increased cytotoxicity. In this study, we compared HePC release of different TSL formulations in serum. HePC showed rapid but incomplete release below the transition temperature (Tm) of investigated TSL formulations in serum. Short heating (5 min) to 42 °C increased HePC release from DPPG2-TSL (Tm = 41 °C) by a factor of two in comparison to body temperature (37 °C). Bovine serum albumin (BSA) induced HePC release from DPPG2-TSL comparable to serum. Furthermore, multilamellar vesicles (MLV) were capable to extract HePC from DPPG2-TSL in a concentration- and temperature-dependent manner. Repetitive exposure of DPPG2-TSL to MLV at 37 °C led to a fast initial release of HePC which slowed down after subsequent extraction cycles finally reaching approx. 50% HePC release. A pharmacokinetic study in rats revealed a biphasic pattern with an immediate clearance of approx. 50% HePC whereas the remaining 50% HePC showed a prolonged circulation time. We speculate that HePC located in the external leaflet of DPPG2-TSL is rapidly released upon contact with suitable biological acceptors. As demonstrated by MLV transfer experiments, asymmetric incorporation of HePC into the internal leaflet of DPPG2-TSL might improve HePC retention in presence of complex biological media and still give rise to HT-induced HePC release.


Assuntos
Antineoplásicos/farmacocinética , Lipossomos , Fosfatidilgliceróis/química , Fosforilcolina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Varredura Diferencial de Calorimetria , Cromatografia Líquida/métodos , Hemólise/efeitos dos fármacos , Humanos , Masculino , Fosforilcolina/farmacocinética , Fosforilcolina/farmacologia , Ratos , Espectrometria de Massas em Tandem/métodos , Temperatura
19.
PLoS Negl Trop Dis ; 15(7): e0009622, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34292975

RESUMO

BACKGROUND: Miltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency. METHODOLOGY/PRINCIPAL FINDINGS: To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production. CONCLUSIONS/SIGNIFICANCE: Differential induction of innate immune responses in the liver was found to underlie the attenuated phenotype of a MIL-R parasite and its peculiar feature of drug-dependency. The impact of MIL on hepatic NK and NKT activation and IFN-γ production following recognition of a MIL-R strain indicates that this mechanism may sustain infections with resistant parasites and contribute to treatment failure.


Assuntos
Resistência a Medicamentos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/patogenicidade , Fosforilcolina/análogos & derivados , Animais , Antiprotozoários/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/genética , Interferon gama/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Leishmaniose Visceral , Fígado/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/fisiologia , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/metabolismo , Neutrófilos , Testes de Sensibilidade Parasitária , Fosforilcolina/farmacologia , Baço/citologia
20.
Parasit Vectors ; 14(1): 366, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34266485

RESUMO

BACKGROUND: Superoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this enzyme is a promising molecular target for drug development against trypanosomatids. RESULTS: We obtained Leishmania infantum mutant clones with lower FeSOD-A expression and investigated their phenotypes. Our attempts to delete this enzyme-coding gene using three different methodologies (conventional allelic replacement or two different CRISPR/methods) failed, as FeSOD-A gene copies were probably retained by aneuploidy or gene amplification. Promastigote forms of WT and mutant parasites were used in quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blot analyses, and these parasite forms were also used to assess drug susceptibility. RT-qPCR and western blot analyses revealed that FeSOD-A transcript and protein levels were lower in FeSOD-A-/-/+ L. infantum mutant clones than in the wild-type (WT) parasite. The decrease in FeSOD-A expression in L. infantum did not interfere with the parasite growth or susceptibility to amphotericin B. Surprisingly, FeSOD-A-/-/+ L. infantum mutant clones were 1.5- to 2.0-fold more resistant to trivalent antimony and 2.4- to 2.7-fold more resistant to miltefosine. To investigate whether the decrease in FeSOD-A expression was compensated by other enzymes, the transcript levels of five FeSODs and six enzymes from the antioxidant defence system were assessed by RT-qPCR. The transcript level of the enzyme ascorbate peroxidase increased in both the FeSOD-A-/-/+ mutants tested. The FeSOD-A-/-/+ mutant parasites were 1.4- to 1.75-fold less tolerant to oxidative stress generated by menadione. Infection analysis using THP-1 macrophages showed that 72 h post-infection, the number of infected macrophages and their intracellular multiplication rate were lower in the FeSOD-A-/-/+ mutant clones than in the WT parasite. CONCLUSIONS: The unsuccessful attempts to delete FeSOD-A suggest that this gene is essential in L. infantum. This enzyme plays an important role in the defence against oxidative stress and infectivity in THP-1 macrophages. FeSOD-A-deficient L. infantum parasites deregulate their metabolic pathways related to antimony and miltefosine resistance.


Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Regulação para Baixo , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Fosforilcolina/análogos & derivados , Superóxido Dismutase/genética , Leishmania infantum/enzimologia , Mutação , Estresse Oxidativo/efeitos dos fármacos , Fosforilcolina/farmacologia
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