Cannabinoid receptor subtype 2 (CB2R) is emerging as a pivotal biomarker to identify the first steps of inflammation-based diseases such as cancer and neurodegeneration. There is an urgent need to ...find specific probes that may result in green and safe alternatives to the commonly used radiative technologies, to deepen the knowledge of the CB2R pathways impacting the onset of the above-mentioned pathologies. Therefore, based on one of the CB2R pharmacophores, we developed a class of fluorescent N-adamantyl-1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives spanning from the green to the near-infrared (NIR) regions of the light spectrum. Among the synthesized fluorescent ligands, the green-emitting compound 55 exhibited a favorable binding profile (strong CB2R affinity and high selectivity). Notably, this ligand demonstrated versatility as its use was validated in different experimental settings such as flow cytometry saturation, competitive fluorescence assays, and in vitro microglia cells mimicking inflammation states where CB2R are overexpressed.Cannabinoid receptor subtype 2 (CB2R) is emerging as a pivotal biomarker to identify the first steps of inflammation-based diseases such as cancer and neurodegeneration. There is an urgent need to find specific probes that may result in green and safe alternatives to the commonly used radiative technologies, to deepen the knowledge of the CB2R pathways impacting the onset of the above-mentioned pathologies. Therefore, based on one of the CB2R pharmacophores, we developed a class of fluorescent N-adamantyl-1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives spanning from the green to the near-infrared (NIR) regions of the light spectrum. Among the synthesized fluorescent ligands, the green-emitting compound 55 exhibited a favorable binding profile (strong CB2R affinity and high selectivity). Notably, this ligand demonstrated versatility as its use was validated in different experimental settings such as flow cytometry saturation, competitive fluorescence assays, and in vitro microglia cells mimicking inflammation states where CB2R are overexpressed.
Some 2,4‐disubstituted quinazolines were synthesized and studied as multidrug resistance (MDR) reversers. The new derivatives carried the quinazoline‐4‐amine scaffold found in modulators of the ABC ...transporters involved in MDR, as the TKIs gefitinib and erlotinib. Their behaviour on the three ABC transporters, P‐gp, MRP1 and BCRP, was investigated. Almost all compounds inhibited the P‐gp activity in MDCK‐MDR1 cells overexpressing P‐gp, showing EC50 values in the nanomolar range (1 d, 1 e, 2 a, 2 c, 2 e). Some compounds were active also towards MRP1 and/or BCRP. Docking results obtained by in silico studies on the P‐gp crystal structure highlighted common features for the most potent compounds. The P‐gp selective compound 1 e was able to increase the doxorubicin uptake in HT29/DX cells and to restore its antineoplastic activity in resistant cancer cells in the same extent of sensitive cells. Compound 2 a displayed a dual inhibitory effect showing good activities towards both P‐gp and BCRP.
2,4‐disubstituted quinazoline derivatives as MDR reversers: Derivatives carrying the quinazoline‐4‐amine scaffold were studied for their activity on ABC transporters, P‐gp, MRP1 and BCRP, involved in multidrug resistance (MDR). Potent P‐gp inhibitors with activity in the nanomolar range were identified. The results allowed us to propose structural requirements for defining P‐gp, MRP1 and BCRP activity and selectivity.
Stereo- and regioisomers of a series of N,N-bis(alkanol)amine aryl ester derivatives have been prepared and studied as multidrug resistance (MDR) modulators. The new compounds contain a ...2-(methyl)propyl chain combined with a 3-, 5- or 7-methylenes long chain and carry different aromatic ester portions. Thus, these compounds have a methyl group on the 3-methylenes chain and represent branched homologues of previously studied derivatives. The introduction of the methyl group gives origin to a stereogenic center and consequently to (R) and (S) enantiomers. In the pirarubicin uptake assay on K562/DOX cell line these compounds showed good activity and efficacy and in many cases enantioselectivity was observed. Docking studies confirmed the influence of the stereocenter on the interaction in the P-gp pocket. The P-gp interaction mechanism and selectivity towards MRP1 and BCRP were also evaluated on MDCK transfected cells overexpressing the three transporters. Almost all these compounds inhibited both P-gp and BCRP, but only derivatives with specific structural characteristics showed MRP1 activity. Moreover, two compounds, (S)-3 and (R)-7, showed the ability to induce collateral sensitivity (CS) against MDR cells. Therefore, these two CS-promoting agents could be considered interesting leads for the development of selective cytotoxic agents for drug-resistant cells.
•Stereo- and regioisomers of amino aryl esters showed different MDR reversing activity profiles.•Compounds (S)-3 (Ar = b, Ar1 = a, n = 3) and (R)-7 (Ar = b, Ar1 = a, n = 5) induced collateral sensitivity (CS) against MDR cells. Display omitted
•Stereo- and regioisomers of N,N-bis(alkanol)amine aryl esters were described.•The biological behavior of these compounds was studied by various tests.•They act as multidrug resistance (MDR) reversers.•Two compounds were identified as MDR1 or MRP1-overexpressing cells sensitizers.•Docking studies and stability tests in human plasma were performed.
The aim of the present work was to compare the mucoadhesive and efflux pump P-glycoprotein (P-gp) interacting properties of chitosan (CS)- and glycolchitosan (GCS)-based thiomers and corresponding ...unmodified parent polymers. For this purpose, the glycol chitosan-N-acetyl-cysteine (GCS-NAC) and glycol chitosan-glutathione (GCS-GSH) thiomers were prepared under simple and mild conditions. Their mucoadhesive characteristics were studied by turbidimetric and zeta potential measurements. The P-gp interacting properties were evaluated measuring the effects of thiolated- and unmodified-polymers on the bidirectional transport (BA/AB) of rhodamine-123 across Caco-2 cells as well as in the calcein-AM and ATPase activity assays. Although all the thiomers and unmodified polymers showed optimal-excellent mucoadhesive properties, the best mucoadhesive performances have been obtained by CS and CS-based thiomers. Moreover, it was found that the pretreatment of Caco-2 cell monolayer with GCS-NAC or GCS restores Rho-123 cell entrance by inhibiting P-gp activity. Hence, GCS-NAC and GCS may constitute new biomaterials useful for improving the bioavailability of P-gp substrates.
1-Cyclohexyl-4-3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propylpiperazine 1 (PB28) and 2-Methoxy-5-methyl-N-4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)butylbenzamide 2 (RHM-1) represent ...leads for tumor diagnosis, given their high affinity at σ(2) receptors. With the purpose of obtaining good candidates for σ(2) PET tracers development, hybrid structures between 1 and 2 were designed. Excellent σ(1)/σ(2) selectivities were reached when 6,7-dimethoxytetrahydroisoquinoline was linked to an o-methoxy substituted arylamide (11a, 12a, 15a), and for these benzamides an intramolecular H-bond in the active conformation at the σ sites, was hypothesized. However these excellent σ(2) ligands were accompanied by interaction with P-gp, which may limit their use as σ(2) receptor PET agents when tumors overexpress P-gp. Compound 15a whose P-gp interaction was just moderate represents an interesting tool for the development of σ(2) PET tracers useful in tumors overexpressing P-gp.
Abstract
BACKGROUND AND AIMS
Emerging evidence suggest that uremic toxins are involved in the progression of chronic kidney disease (CKD). Indoxyl Sulfate (IxS) is one of the most studied since ...resulted as strong predictor of cardiovascular disease (CVD) and linked with the prognosis of patients with CKD. Indeed, blood levels of IxS increase during CKD stages reaching a maximum in patients with end-stage renal disease (ESRD). Additionally, hemodialysis is able to eliminate only the blood-free fraction as the IxS albumin bond hides its removal. As a result, patients on dialysis show the highest IxS levels resulting in worsening condition, enhancement of CVD-related complication. Thus, IxS rapid quantification could offers clinicians an alternative method for monitoring CKD progression and address the dialysis effectiveness. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is commonly used for IxS detection and require dedicated equipment and skills, long analysis times and costly analytical instruments. To address these issues, the development of a simple and rapid analytical method for IxS detection is needed. Here, we present a study focused on the development and validation of a rapid and simple method of IxS determination based on colorimetric array-based analysis exploitable in clinical practice for CKD patients.
METHOD
A total of 20 blood samples (10 healthy people and 10 CKD patients) were used to assess the feasibility of our colorimetric assay. IxS from serum sample was quantified at time of inclusion by LC-MS/MS. Next, from the same samples, we used a derivatization reaction for splitting the serum IxS into indigo-blue. This method enables the rapid quantification of IxS-derived indigo by a smart spectrophotometer at λ = 420–450 nm. Analyte concentration was instantly calculated from absorbance/concentration calibration curve of indigo-blue. Finally, a correlation between the two methods were carried out for the colorimetric analysis validation.
RESULTS
To accurately determine concentrations of IxS in the low µM range for practical diagnostic purposes, a wide linear calibration curve of was successfully established. The calibration curve ranged from ‘0 µM to 330 µM’ for indigo blue. The regression equation between absorbance and concentration was y = 0.0003 x + 0.0192, and the correlation coefficient (r2) was 0.9941. The feasibility was confirmed by detecting IxS in serum samples from healthy people and CKD patient. A positive correlation was observed between IxS quantified by LC-MS/MS and IxS-derived indigo quantified by spectrophotometry (P < 0 001, r = 0.088). Furthermore, the results of colorimetric analysis were compared with those obtained using LC–MS/MS, and no significant difference was found between them (Fig. 2). This result indicated that the colorimetric detection of IxS is comparable to LC–MS/MS, but it is simpler and faster for CKD monitoring in clinical practice.
CONCLUSION
The aim of our study was to develop and validate a quantitative method for the rapid estimation of serum IxS employable to monitor CKD progression in clinical setting, identify patient at risk and help further dialysis therapies aimed to lowering blood uremic toxins. Conventionally, LC-MS/MS is the most used diagnostic tool for the IxS quantification due to high sensitivity and accuracy. However, the great rigorousness analysis together with the requirement of real-time results hinders its employment in the clinical practice. Our results provide a low-cost, easy-to-operate, label-free, rapid and sensitive method for IxS determination for application in tracking IxS in blood and studying CKD progression.
Cannabinoid type 2 receptor (CB2R) is a G-protein-coupled receptor that, together with Cannabinoid type 1 receptor (CB1R), endogenous cannabinoids and enzymes responsible for their synthesis and ...degradation, forms the EndoCannabinoid System (ECS). In the last decade, several studies have shown that CB2R is overexpressed in activated central nervous system (CNS) microglia cells, in disorders based on an inflammatory state, such as neurodegenerative diseases, neuropathic pain, and cancer. For this reason, the anti-inflammatory and immune-modulatory potentials of CB2R ligands are emerging as a novel therapeutic approach. The design of selective ligands is however hampered by the high sequence homology of transmembrane domains of CB1R and CB2R. Based on a recent three-arm pharmacophore hypothesis and latest CB2R crystal structures, we designed, synthesized, and evaluated a series of new N-adamantyl-anthranil amide derivatives as CB2R selective ligands. Interestingly, this new class of compounds displayed a high affinity for human CB2R along with an excellent selectivity respect to CB1R. In this respect, compounds exhibiting the best pharmacodynamic profile in terms of CB2R affinity were also evaluated for the functional behavior and molecular docking simulations provided a sound rationale by highlighting the relevance of the arm 1 substitution to prompt CB2R action. Moreover, the modulation of the pro- and anti-inflammatory cytokines production was also investigated to exert the ability of the best compounds to modulate the inflammatory cascade.
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A series of derivatives were synthesized and studied with the aim to investigate the structure-activity relationships of the two P-glycoprotein (P-gp) modulators elacridar and tariquidar. Then, ...different aryl-substituted amides were inserted, and to explore the effects of varying the amide function, the corresponding isosteric ester derivatives and some alkylamine analogues were synthesized. The new compounds were studied to evaluate their P-gp interaction profile and selectivity toward the two other ABC transporters, multidrug-resistance-associated protein-1 (MRP-1) and breast cancer resistance protein (BCRP). Investigation of the chemical stability of the amide and ester derivatives toward spontaneous or enzymatic hydrolysis showed that these compounds were stable in phosphate-buffered saline and human plasma. This study allowed us to evaluate the selectivity of the three series on the three efflux pumps and to propose the structural requirements that define the P-gp interaction profile. We identified two P-gp substrates, a P-gp inhibitor, and three ester derivatives that were active on BCRP, which opens a new scenario in the development of ligands active toward this pump.
PB183, a PET probe suitable in prostate adenocarcinoma.
PB183, a non-selective sigma receptor ligand displaying high sigma-1 and sigma-2 receptor affinity, was evaluated in prostate tumour cell lines ...for its suitability as PET radiotracer. The pharmacodynamic and pharmacokinetic properties suggested
PB183 as a potential PET radiotracer to visualize prostate adenocarcinoma.
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