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1.
Molecules ; 29(13)2024 Jun 23.
Article in English | MEDLINE | ID: mdl-38998940

ABSTRACT

Aryl Hydrocarbon Receptor (AHR) ligands, upon binding, induce distinct gene expression profiles orchestrated by the AHR, leading to a spectrum of pro- or anti-inflammatory effects. In this study, we designed, synthesized and evaluated three indole-containing potential AHR ligands (FluoAHRL: AGT-4, AGT-5 and AGT-6). All synthesized compounds were shown to emit fluorescence in the near-infrared. Their AHR agonist activity was first predicted using in silico docking studies, and then confirmed using AHR luciferase reporter cell lines. FluoAHRLs were tested in vitro using mouse peritoneal macrophages and T lymphocytes to assess their immunomodulatory properties. We then focused on AGT-5, as it illustrated the predominant anti-inflammatory effects. Notably, AGT-5 demonstrated the ability to foster anti-inflammatory regulatory T cells (Treg) while suppressing pro-inflammatory T helper (Th)17 cells in vitro. AGT-5 actively induced Treg differentiation from naïve CD4+ cells, and promoted Treg proliferation, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expression and interleukin-10 (IL-10) production. The increase in IL-10 correlated with an upregulation of Signal Transducer and Activator of Transcription 3 (STAT3) expression. Importantly, the Treg-inducing effect of AGT-5 was also observed in human tonsil cells in vitro. AGT-5 showed no toxicity when applied to zebrafish embryos and was therefore considered safe for animal studies. Following oral administration to C57BL/6 mice, AGT-5 significantly upregulated Treg while downregulating pro-inflammatory Th1 cells in the mesenteric lymph nodes. Due to its fluorescent properties, AGT-5 could be visualized both in vitro (during uptake by macrophages) and ex vivo (within the lamina propria of the small intestine). These findings make AGT-5 a promising candidate for further exploration in the treatment of inflammatory and autoimmune diseases.


Subject(s)
Receptors, Aryl Hydrocarbon , T-Lymphocytes, Regulatory , Animals , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Aryl Hydrocarbon/agonists , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , Mice , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/chemical synthesis , Humans , Zebrafish , Fluorescent Dyes/chemistry , Ligands , Mice, Inbred C57BL , Indoles/pharmacology , Indoles/chemistry , Cell Differentiation/drug effects
2.
Molecules ; 28(5)2023 Mar 02.
Article in English | MEDLINE | ID: mdl-36903557

ABSTRACT

The simultaneous administration of three antiplatelet agents has been proposed as an efficient strategy for the secondary prevention of atherothrombotic events and is included in the European guidelines. However, this strategy presented an increased risk of bleeding; therefore, the identification of new antiplatelet agents, with improved efficacy and diminished side effects, is of great importance. In silico studies, UPLC/MS Q-TOF plasma stability, in vitro platelet aggregation experiments, and pharmacokinetic studies were exploited. In the present study, it has been predicted that the flavonoid apigenin could target different platelet activation pathways, including P2Y12, protease-activated receptor-1 (PAR-1), and cyclooxygenase 1 (COX-1). To enhance apigenin's potency, hybridization with docosahexaenoic acid (DHA) was performed, as fatty acids have illustrated potent efficacy against cardiovascular diseases (CVDs). The new molecular hybrid, termed 4'-DHA-apigenin, demonstrated enhanced inhibitory activity against platelet aggregation induced by thrombin receptor activator peptide-6 (TRAP-6), adenosine diphosphate (ADP), and arachidonic acid (AA), with respect to the parent apigenin. The 4'-DHA-apigenin hybrid illustrated an almost 2-fold enhanced inhibitory activity, with respect to apigenin, and an almost 3-fold enhanced inhibitory activity, with respect to DHA, for the ADP-induced platelet aggregation. Additionally, the hybrid presented a more than 12-fold enhanced inhibitory activity with respect to DHA for the TRAP-6 induced platelet aggregation. Furthermore, a 2-fold enhanced inhibitory activity was recorded for the 4'-DHA-apigenin hybrid for the AA-induced platelet aggregation with respect to apigenin. To surmount the reduced LC-MS based plasma stability, a novel dosage form in olive oil has been developed. The 4'-DHA-apigenin olive oil-based formulation presented an enhanced antiplatelet inhibitory effect in three activation pathways. To further explore the pharmacokinetic profile of 4'-DHA-apigenin in olive oil formulations, a UPLC/MS Q-TOF protocol has been established to quantify the serum levels of apigenin after oral administration to C57BL/6J wild type mice. The olive oil-based formulation of 4'-DHA-apigenin demonstrated an increase in apigenin bioavailability of 262 %. This study may offer a new therapeutic strategy tailored to improve the treatment of CVDs.


Subject(s)
Cardiovascular Diseases , Platelet Aggregation Inhibitors , Animals , Mice , Platelet Aggregation Inhibitors/pharmacology , Apigenin/pharmacology , Fibrinolytic Agents/pharmacology , Olive Oil/pharmacology , Mice, Inbred C57BL , Platelet Aggregation , Cardiovascular Diseases/drug therapy , Arachidonic Acid/pharmacology , Adenosine Diphosphate/pharmacology
3.
Plant Foods Hum Nutr ; 77(2): 198-205, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35397767

ABSTRACT

Replacing synthetic dyes with natural pigments has gained great attention over the past years in the food industry, due to the increased alertness of consumers for nontoxic and natural additives. Betalains are water-soluble nitrogenous natural pigments that are used as natural colorants in food industries, due to their applicability and their rich pharmacological profile including antioxidant, antimicrobial, and anticancer properties. Therefore, there is a need for a detailed exploration of betalains to fully exploit their properties. Opuntia spp. plants are one of the primary sources of betalains. The objective of this study was to identify betalain phytochemical content in prickly pear cactus of two different Opuntia species from Greece (an Opuntia ficus-indica (L.) Mill (OFI) orange prickly pear cultivar and an Opuntia spp. purple prickly pear cultivar) using modern analytical techniques as also to evaluate their antioxidant and cytotoxicity profile. To achieve this we used an array of analytical techniques, including ultra-violet-vis (UV-Vis) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) as also cell based in vitro assays. These enabled us to establish a rapid approach that can distinguish the different Opuntia spp. cultivars based on their phytochemical constituents through untargeted metabolomics analysis using ultra-high performance liquid chromatography-mass spectrometry - quadrupole time-of-flight (UPLC/MS Q-TOF). These findings could allow a further exploitation of Opuntia species and especially their enriched betalain phytochemical profile as viable source of natural food colorants.


Subject(s)
Citrus sinensis , Opuntia , Antioxidants/analysis , Betalains/analysis , Betalains/chemistry , Betalains/pharmacology , Fruit/chemistry , Greece , Opuntia/chemistry , Phytochemicals/analysis
4.
Molecules ; 27(1)2021 Dec 23.
Article in English | MEDLINE | ID: mdl-35011300

ABSTRACT

Biobased pigments are environmentally friendly alternatives to synthetic variants with an increased market demand. Production of pigments via fermentation is a promising process, yet optimization of the production yield and rate is crucial. Herein, we evaluated the potential of Penicillium purpurogenum to produce biobased pigments. Optimum sugar concentration was 30 g/L and optimum C:N ratio was 36:1 resulting in the production of 4.1-4.5 AU (namely Pigment Complex A). Supplementation with ammonium nitrate resulted in the production of 4.1-4.9 AU (namely Pigment Complex B). Pigments showed excellent pH stability. The major biopigments in Pigment Complex A were N-threonyl-rubropunctamin or the acid form of PP-R (red pigment), N-GABA-PP-V (violet pigment), PP-O (orange pigment) and monascorubrin. In Pigment Complex B, a novel biopigment annotated as N-GLA-PP-V was identified. Its basic structure contains a polyketide azaphilone with the same carboxyl-monascorubramine base structure as PP-V (violet pigment) and γ-carboxyglutamic acid (GLA). The pigments were not cytotoxic up to 250 µg/mL.


Subject(s)
Biological Products/chemistry , Biological Products/pharmacology , Drug Discovery , Penicillium/chemistry , Pigments, Biological/chemistry , Pigments, Biological/pharmacology , Antioxidants/chemistry , Antioxidants/pharmacology , Biological Products/isolation & purification , Carbon/chemistry , Cell Survival/drug effects , Drug Discovery/methods , Fermentation , Glucose/chemistry , Humans , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Mass Spectrometry , Nitrogen/chemistry , Penicillium/metabolism , Pigments, Biological/isolation & purification , Spectrum Analysis
5.
Methods Mol Biol ; 2207: 235-246, 2021.
Article in English | MEDLINE | ID: mdl-33113140

ABSTRACT

Many bioactive substances face the problem of limited bioavailability, mainly due to low aqueous solubility and poor metabolic stability. Their complexation with drug delivery systems offers a more optimum pharmacological profile. Some of these drug delivery systems that have promising potential form complexes with bioactive compounds such as cyclodextrins and calixarenes. The monitoring of the success and the type of the complexation are of great importance and two-dimensional diffusion-ordered NMR spectroscopy (2D DOSY) is a valuable tool for the studying of these complexes and described as "NMR chromatography." Herein we report the procedure for the complexation of the natural product quercetin in 2-hydroxypropyl-ß-cyclodextrin and the anticancer drug temozolomide in p-sulfonatocalix[4]arene and the determination of the complexation with 2D DOSY spectroscopy.


Subject(s)
2-Hydroxypropyl-beta-cyclodextrin/chemistry , Drug Carriers/chemistry , Nuclear Magnetic Resonance, Biomolecular , Temozolomide/chemistry
6.
Eur J Med Chem ; 166: 256-266, 2019 Mar 15.
Article in English | MEDLINE | ID: mdl-30716713

ABSTRACT

Peptide-drug conjugates have emerged as a potent approach to enhance the targeting and pharmacokinetic profiles of drugs. However, the impact of the linker unit has not been explored/exploited in depth. Gemcitabine (dFdC) is an anticancer agent used against a variety of solid tumours. Despite its potency, gemcitabine suffers mostly due to its unspecific toxicity, lack of targeting and rapid metabolic inactivation. To minimize these limitations and enable its targeting to tumours overexpressing the GnRH receptor, we examined the peptide-drug conjugation approach. Our design hypothesis was driven by the impact that the linker unit could have on the peptide-drug conjugate efficacy. Along these lines, in order to exploit the potential to manipulate the potency of gemcitabine through altering the linker unit we constructed three different novel peptide-drug conjugates assembled of gemcitabine, the tumour-homing peptide D-Lys6-GnRH and modified linker building blocks. Specifically, the linker was sculpted to either allow slow drug release (utilizing carbamate bond) or rapid disassociation (using amide and ester bonds). Notably, the new analogues possessed up to 95.5-fold enhanced binding affinity for the GnRH receptor (GnRH-R) compared to the natural peptide ligand D-Lys6-GnRH. Additionally, their in vitro cytotoxicity was evaluated in four different cancer cell lines. Their cellular uptake, release of gemcitabine and inactivation of gemcitabine to its inactive metabolite (dFdU) was explored in a representative cell line. In vitro stability and the consequent drug release were evaluated in cell culture medium and human plasma. In vivo pharmacokinetic studies were performed in mice, summarizing the relative stability of the three conjugates and the released levels of gemcitabine in comparison with dFdU. These studies suggest that the fine tuning of the linkage within a peptide-drug conjugate affects the drug release rate and its overall pharmaceutical profile. This could eventually emerge as an intriguing medicinal chemistry approach to optimize bio-profiles of prodrugs.


Subject(s)
Deoxycytidine/analogs & derivatives , Drug Liberation , Gonadotropin-Releasing Hormone/chemistry , Lysine/chemistry , Prodrugs/metabolism , Animals , Cell Proliferation/drug effects , Deoxycytidine/chemistry , Deoxycytidine/metabolism , Deoxycytidine/pharmacokinetics , Deoxycytidine/pharmacology , Drug Stability , Humans , Intracellular Space/metabolism , MCF-7 Cells , Mice , Receptors, LHRH/metabolism , Gemcitabine
7.
Mol Pharm ; 16(3): 1255-1271, 2019 03 04.
Article in English | MEDLINE | ID: mdl-30681344

ABSTRACT

Renin-angiotensin aldosterone system inhibitors are for a long time extensively used for the treatment of cardiovascular and renal diseases. AT1 receptor blockers (ARBs or sartans) act as antihypertensive drugs by blocking the octapeptide hormone Angiotensin II to stimulate AT1 receptors. The antihypertensive drug candesartan (CAN) is the active metabolite of candesartan cilexetil (Atacand, CC). Complexes of candesartan and candesartan cilexetil with 2-hydroxylpropyl-ß-cyclodextrin (2-HP-ß-CD) were characterized using high-resolution electrospray ionization mass spectrometry and solid state 13C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy. The 13C CP/MAS results showed broad peaks especially in the aromatic region, thus confirming the strong interactions between cyclodextrin and drugs. This experimental evidence was in accordance with molecular dynamics simulations and quantum mechanical calculations. The synthesized and characterized complexes were evaluated biologically in vitro. It was shown that as a result of CAN's complexation, CAN exerts higher antagonistic activity than CC. Therefore, a formulation of CC with 2-HP-ß-CD is not indicated, while the formulation with CAN is promising and needs further investigation. This intriguing result is justified by the binding free energy calculations, which predicted efficient CC binding to 2-HP-ß-CD, and thus, the molecule's availability for release and action on the target is diminished. In contrast, CAN binding was not favored, and this may allow easy release for the drug to exert its bioactivity.


Subject(s)
2-Hydroxypropyl-beta-cyclodextrin/chemistry , Angiotensin II Type 1 Receptor Blockers/chemistry , Benzimidazoles/chemistry , Biphenyl Compounds/chemistry , Drug Compounding/methods , Prodrugs/chemistry , Tetrazoles/chemistry , Adaptor Proteins, Signal Transducing/chemistry , Benzimidazoles/chemical synthesis , Carbon-13 Magnetic Resonance Spectroscopy , HEK293 Cells , Humans , Hydrogen Bonding , Molecular Conformation , Molecular Dynamics Simulation , Renin-Angiotensin System , Spectrometry, Fluorescence , Spectrometry, Mass, Electrospray Ionization , Tetrazoles/chemical synthesis
8.
Biochim Biophys Acta Gen Subj ; 1862(9): 1913-1924, 2018 09.
Article in English | MEDLINE | ID: mdl-29886278

ABSTRACT

BACKGROUND: Flavonoids possess a rich polypharmacological profile and their biological role is linked to their oxidation state protecting DNA from oxidative stress damage. However, their bioavailability is hampered due to their poor aqueous solubility. This can be surpassed through encapsulation to supramolecular carriers as cyclodextrin (CD). A quercetin- 2HP-ß-CD complex has been formerly reported by us. However, once the flavonoid is in its 2HP-ß-CD encapsulated state its oxidation potential, its decomplexation mechanism, its potential to protect DNA damage from oxidative stress remained elusive. To unveil this, an array of biophysical techniques was used. METHODS: The quercetin-2HP-ß-CD complex was evaluated through solubility and dissolution experiments, electrochemical and spectroelectrochemical studies (Cyclic Voltammetry), UV-Vis spectroscopy, HPLC-ESI-MS/MS and HPLC-DAD, fluorescence spectroscopy, NMR Spectroscopy, theoretical calculations (density functional theory (DFT)) and biological evaluation of the protection offered against H2O2-induced DNA damage. RESULTS: Encapsulation of quercetin inside the supramolecule's cavity enhanced its solubility and retained its oxidation profile. Although the protective ability of the quercetin-2HP-ß-CD complex against H2O2 was diminished, iron serves as a chemical stimulus to dissociate the complex and release quercetin. CONCLUSIONS: We found that in a quercetin-2HP-ß-CD inclusion complex quercetin retains its oxidation profile similarly to its native state, while iron can operate as a chemical stimulus to release quercetin from its host cavity. GENERAL SIGNIFICANCE: The oxidation profile of a natural product once it is encapsulated in a supramolecular carrier was unveiled as also it was discovered that decomplexation can be triggered by a chemical stimilus.


Subject(s)
Cyclodextrins/metabolism , DNA Damage/drug effects , Hydrogen Peroxide/pharmacology , Iron/metabolism , Quercetin/metabolism , Biological Availability , Cyclodextrins/chemistry , Humans , Iron/chemistry , Jurkat Cells , Oxidants/pharmacology , Oxidation-Reduction , Oxidative Stress/drug effects , Quercetin/chemistry
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