Development of a Novel Apigenin Dosage form as a Substitute for the Modern Triple Antithrombotic Regimen.

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.

Exploration of Betalains and Determination of the Antioxidant and Cytotoxicity Profile of Orange and Purple Opuntia spp. Cultivars in Greece.

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.

Interplay of cholesterol, membrane bilayers and the AT1R: A cholesterol consensus motif on AT1R is revealed.

Hypertension, mediated by the Angiotensin II receptor type 1 (AT1R), is still the major cause of premature death despite the discovery of novel therapeutics, highlighting the importance of an in depth understanding of the drug-AT1R recognition mechanisms coupled with the impact of the membrane environment on the interaction of drugs with AT1R. Herein, we examine the interplay of cholesterol-lipid-candesartan and the AT1R using Molecular Dynamics simulations of a model membrane consisting of 60:40 mol%. DPPC:cholesterol, candesartan and the AT1R, mimicking the physiological cholesterol concentration in sarcolemma membranes. The simulations of the model membrane of 60:40 mol%. DPPC:cholesterol were further validated using DOSY NMR experiments. Interestingly, our results suggest a significant role of cholesterol in the AT1R function imposed through a Cholesterol Consensus Motif (CCM) in the receptor, which could be crucial in the drug binding process. Candesartan diffusion towards AT1R through incorporation into lipid bilayers, appears to be retarded by the presence of cholesterol. However, its direct approach towards AT1R may be facilitated through the mobility induced on the N-terminus by the cholesterol binding on the CCM these novel insights could pave the way towards the development of more potent pharmaceutical agents to combat hypertension more effectively.

Chemical Profiling, Bioactivity Evaluation and the Discovery of a Novel Biopigment Produced by Penicillium purpurogenum CBS 113139.

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.

2D DOSY NMR: A Valuable Tool to Confirm the Complexation in Drug Delivery Systems.

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.

Unveiling the interaction profile of rosmarinic acid and its bioactive substructures with serum albumin.

Rosmarinic acid, a phytochemical compound, bears diverse pharmaceutical profile. It is composed by two building blocks: caffeic acid and a salvianic acid unit. The interaction profile, responsible for the delivery of rosmarinic acid and its two substructure components by serum albumin remains unexplored. To unveil this, we established a novel low-cost and efficient method to produce salvianic acid from the parent compound. To probe the interaction profile of rosmarinic acid and its two substructure constituents with the different serum albumin binding sites we utilised fluorescence spectroscopy and competitive saturation transfer difference NMR experiments. These studies were complemented with transfer NOESY NMR experiments. The thermodynamics of the binding profile of rosmarinic acid and its substructures were addressed using isothermal titration calorimetry. In silico docking studies, driven by the experimental data, have been used to deliver further atomic details on the binding mode of rosmarinic acid and its structural components.

Exploring the role of the membrane bilayer in the recognition of candesartan by its GPCR AT1 receptor.

Cardiovascular diseases and hypertension in particular are major health risks worldwide and the improvement on their treatment will be beneficial for the human health. AT1R antagonists belong to the sartans family that targets the renin-angiotensin aldosterone system (RAAS) through blocking the hormone angiotensin II to exert its detrimental effects in pathological states. As a consequence, they are beneficial to treat hypertension, diabetes related kidney failure and hyperaemic episodes. Long unbiased Molecular Dynamics (MD) simulations are performed in order to explore candesartan's possible 2D and 3D diffusion mechanisms towards AT1R receptor. 3D diffusion mechanism is referred to the direct binding of the AT1 antagonist candesartan to the AT1R 3D structure (PDB ID: 4YAY). 2D diffusion mechanism involves first, the incorporation of candesartan in the bilayer core and then its localization on the AT1R binding cavity, through a diffusion mechanism. The obtained results indicate that membranes interact significantly with the neutral form of candesartan, which is indeed approaching the receptors' active site through diffusion via the lipids. On the other hand, the deprotonated form of the drug is interacting with AT1R's extracellular loop and fails to enter the membrane, pointing out the importance of the pH microenvironment around the receptor. To validate the calculated diffusion coefficients of the drug in the lipid bilayers 2D DOSY NMR experiments were recorded and they were in good agreement. Information on the impact that has the interaction of candesartan with the membrane is very important for the rationally design and development of potent ARBs. Thus, its conformational features as well as its localization in the membrane core have to be thoroughly explored.

Encapsulation of Temozolomide in a Calixarene Nanocapsule Improves Its Stability and Enhances Its Therapeutic Efficacy against Glioblastoma.

The alkylating agent temozolomide (TMZ) is the first-line chemotherapeutic for glioblastoma (GBM), a common and aggressive primary brain tumor in adults. However, its poor stability and unfavorable pharmacokinetic profile limit its clinical efficacy. There is an unmet need to tailor the therapeutic window of TMZ, either through complex derivatization or by utilizing pharmaceutical excipients. To enhance stability and aqueous solubility, we encapsulated TMZ in a p-sulphonatocalix[4]arene (Calix) nanocapsule and used 1H-NMR, LC-MS, and UV-Vis spectroscopy to chart the stability of this novel TMZ@Calix complex according to FDA and European Medicines Agency guidelines. LC-MS/MS plasma stability assays were conducted in mice to further explore the stability profile of TMZ@Calix in vivo The therapeutic efficacy of TMZ@Calix was compared with that of unbound TMZ in GBM cell lines and patient-derived primary cells with known O6-methylguanine-DNA methyltransferase (MGMT) expression status and in vivo in an intracranial U87 xenograft mouse model. Encapsulation significantly enhanced the stability of TMZ in all conditions tested. TMZ@Calix was more potent than native TMZ at inhibiting the growth of established GBM cell lines and patient-derived primary lines expressing MGMT and highly resistant to TMZ. In vivo, native TMZ was rapidly degraded in mouse plasma, whereas the stability of TMZ@Calix was enhanced threefold with increased therapeutic efficacy in an orthotopic model. In the absence of new effective therapies, this novel formulation is of clinical importance, serving as an inexpensive and highly efficient treatment that could be made readily available to patients with GBM and warrants further preclinical and clinical evaluation.

Development of bioactive gemcitabine-D-Lys6-GnRH prodrugs with linker-controllable drug release rate and enhanced biopharmaceutical profile.

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.

Host-Guest Interactions between Candesartan and Its Prodrug Candesartan Cilexetil in Complex with 2-Hydroxypropyl-ß-cyclodextrin: On the Biological Potency for Angiotensin II Antagonism.

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.

Exploring the oxidation and iron binding profile of a cyclodextrin encapsulated quercetin complex unveiled a controlled complex dissociation through a chemical stimulus.

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.

Dose perturbation in the radiotherapy of breast cancer patients implanted with the Magna-Site: a Monte Carlo study.

External beam radiation therapy (RT) is often offered to breast cancer patients after surgical mastectomy followed by breast reconstruction with silicone implants. In some cases, the RT is administered while the patient is still implanted with a temporary tissue expander including a high-density metallic port, which is expected to affect the planned dose distribution. This work uses Monte Carlo (MC) simulation in order to evaluate the aforementioned effect when the McGhan Style 133 Tissue Expander with the Magna-Site injection port is used. Simulations have been performed on a patient model built using the actual CT images of the patient for two irradiation schemes, involving two tangential photon beams of 6 MV and 18 MV respectively. MC results show that the presence of the Magna-Site within the two irradiation fields leads to an overall reduction of absorbed dose for points lying in the shadow of the metallic port (relative to each of the opposing beams). The relative reduction compared to dose results without the expander in place ranges from 7% to 13% for the 6 MV beam and is around 6% for the 18 MV photon beam. However, in the close vicinity of the metallic port, increased absorbed doses are observed, due to the increase of secondary electrons emerging from the metallic part of the insert.

[Absorption dose in scintimammography and in X-rays mammography].

Absorbed dose calculations in diagnostic applications using ionizing radiation are more accurate today than in past times. In this work we aim to demonstrate the methods used to calculate the absorbed dose of X-rays mammography and scintimammography. Absorbed dose estimation is achieved by calculations for internal dosimetry using Medical Internal Radiation Dose (MIRD), or Monte Carlo techniques, measurements in phantoms for mammography as well as patient specific calculations exploiting scintigraphic images' data. Especially, the accurate calculation of the absorbed doses during diagnostic examinations of the breast gives the possibility of evaluation of the danger of the use of ionizing radiation for this organ. Optimization of used techniques points to the reduction of the radiation burden of the examined person, by these screening tests. As the radio sensitivity of breast is high, the selection of an accurate absorbed dose calculation method is necessary. The weighting factor for breast is 0.12. This is the maximum value of the weighting factor for the various organs of the human body. Consequently, a detailed study of absorbed dose in breast, either in mammography or scintimammography, is crucial.