Conjugating uncoupler compounds with hydrophobic hydrocarbon chains to achieve adipose tissue selective drug accumulation.

One potential approach for treating obesity is to increase energy expenditure in brown and white adipose tissue. Here we aimed to achieve this outcome by targeting mitochondrial uncoupler compounds selectively to adipose tissue, thus avoiding side effects from uncoupling in other tissues. Selective drug accumulation in adipose tissue has been observed with many lipophilic compounds and dyes. Hence, we explored the feasibility of conjugating uncoupler compounds with a lipophilic C8-hydrocarbon chain via an ether bond. We found that substituting the trifluoromethoxy group in the uncoupler FCCP with a C8-hydrocarbon chain resulted in potent uncoupling activity. Nonetheless, the compound did not elicit therapeutic effects in mice, likely as a consequence of metabolic instability resulting from rapid ether bond cleavage. A lipophilic analog of the uncoupler compound 2,6-dinitrophenol, in which a C8-hydrocarbon chain was conjugated via an ether bond in the para-position (2,6-dinitro-4-(octyloxy)phenol), exhibited increased uncoupling activity compared to the parent compound. However, in vivo pharmacokinetics studies suggested that 2,6-dinitro-4-(octyloxy)phenol was also metabolically unstable. In conclusion, conjugation of a hydrophobic hydrocarbon chain to uncoupler compounds resulted in sustained or improved uncoupling activity. However, an ether bond linkage led to metabolic instability, indicating the need to conjugate lipophilic groups via other chemical bonds.

Cell-derived nanovesicles from mesenchymal stem cells as extracellular vesicle-mimetics in wound healing.

Wound healing is a dynamic process that involves a series of molecular and cellular events aimed at replacing devitalized and missing cellular components and/or tissue layers. Recently, extracellular vesicles (EVs), naturally cell-secreted lipid membrane-bound vesicles laden with biological cargos including proteins, lipids, and nucleic acids, have drawn wide attention due to their ability to promote wound healing and tissue regeneration. However, current exploitation of EVs as therapeutic agents is limited by their low isolation yields and tedious isolation processes. To circumvent these challenges, bioinspired cell-derived nanovesicles (CDNs) that mimic EVs were obtained by shearing mesenchymal stem cells (MSCs) through membranes with different pore sizes. Physical characterisations and high-throughput proteomics confirmed that MSC-CDNs mimicked MSC-EVs. Moreover, these MSC-CDNs were efficiently uptaken by human dermal fibroblasts and demonstrated a dose-dependent activation of MAPK signalling pathway, resulting in enhancement of cell proliferation, cell migration, secretion of growth factors and extracellular matrix proteins, which all promoted tissue regeneration. Of note, MSC-CDNs enhanced angiogenesis in human dermal microvascular endothelial cells in a 3D PEG-fibrin scaffold and animal model, accelerating wound healing in vitro and in vivo. These findings suggest that MSC-CDNs could replace both whole cells and EVs in promoting wound healing and tissue regeneration.

Site-directed deuteration of dronedarone preserves cytochrome P4502J2 activity and mitigates its cardiac adverse effects in canine arrhythmic hearts.

Cytochrome P4502J2 (CYP2J2) metabolizes arachidonic acid (AA) to cardioprotective epoxyeicosatrienoic acids (EETs). Dronedarone, an antiarrhythmic drug prescribed for treatment of atrial fibrillation (AF) induces cardiac adverse effects (AEs) with poorly understood mechanisms. We previously demonstrated that dronedarone inactivates CYP2J2 potently and irreversibly, disrupts AA-EET pathway leading to cardiac mitochondrial toxicity rescuable via EET enrichment. In this study, we investigated if mitigation of CYP2J2 inhibition prevents dronedarone-induced cardiac AEs. We first synthesized a deuterated analogue of dronedarone (termed poyendarone) and demonstrated that it neither inactivates CYP2J2, disrupts AA-EETs metabolism nor causes cardiac mitochondrial toxicity in vitro. Our patch-clamp experiments demonstrated that pharmacoelectrophysiology of dronedarone is unaffected by deuteration. Next, we show that dronedarone treatment or CYP2J2 knockdown in spontaneously beating cardiomyocytes indicative of depleted CYP2J2 activity exacerbates beat-to-beat (BTB) variability reflective of proarrhythmic phenotype. In contrast, poyendarone treatment yields significantly lower BTB variability compared to dronedarone in cardiomyocytes indicative of preserved CYP2J2 activity. Importantly, poyendarone and dronedarone display similar antiarrhythmic properties in the canine model of persistent AF, while poyendarone substantially reduces beat-to-beat variability of repolarization duration suggestive of diminished proarrhythmic risk. Our findings prove that deuteration of dronedarone prevents CYP2J2 inactivation and mitigates dronedarone-induced cardiac AEs.

Rational deuteration of dronedarone attenuates its toxicity in human hepatic HepG2 cells.

Deuteration is a chemical modification strategy that has recently gained traction in drug development. The replacement of one or more hydrogen atom(s) in a drug molecule with its heavier stable isotope deuterium can enhance its metabolic stability and pharmacokinetic properties. However, it remains uninterrogated if rational deuteration at bioactivation "hot-spots" could attenuate its associated toxicological consequences. Here, our preliminary screening with benzofuran antiarrhythmic agents first revealed that dronedarone and its major metabolite N-desbutyldronedarone elicited a greater loss of viability and cytotoxicity in human hepatoma G2 (HepG2) cells as compared with amiodarone and its corresponding metabolite N-desethylamiodarone. A comparison of dronedarone and its in-house synthesized deuterated analogue (termed poyendarone) demonstrated that deuteration could attenuate its in vitro toxicity in HepG2 cells by modulating the extent of mitochondrial dysfunction, reducing the dissipation of mitochondrial membrane potential, and evoking a distinct apoptotic kinetic signature. Furthermore, although pretreatment with the CYP3A inducer rifampicin or the substitution of glucose with galactose in the growth media significantly augmented the loss of cell viability elicited by dronedarone and poyendarone, a lower loss of cell viability was consistently observed in poyendarone across all concentrations. Taken together, our preliminary investigations suggested that the rational deuteration of dronedarone at its benzofuran ring reduces aberrant cytochrome P450 3A4/5-mediated bioactivation, which attenuated its mitochondrial toxicity in human hepatic HepG2 cells.

Enhanced skin penetration of berberine from proniosome gel attenuates pain and inflammation in a mouse model of osteoarthritis.

Dermal delivery of bioactive molecules remains an attractive route of administration in osteoarthritis (OA) due to the local accumulation of drugs while avoiding their systemic side effects. In this study we propose a proniosome gel comprising non-ionic surfactants that self-assemble into de-hydrated vesicles for the delivery of the natural anti-inflammatory compound berberine. By modulating the hydrating ability of the proniosome gel, berberine can be efficiently released with minimal mechanical force. A combination of sorbitan oleate (S80) and polyethlene glycol sorbitan monolaurate (T20) in a sorbitan stearate (S60)-based proniosome enables a readily hydrated gel to deliver berberine into the skin, as confirmed by ex vivo skin permeation studies. Concurrently, an in vitro model of OA using primary mouse chondrocytes demonstrated that the release of berberine at a concentration as low as 1 µg mL-1 is sufficient to restore the production of sulphated glycosaminoglycans (sGAG) to levels comparable to healthy chondrocytes while avoiding the cytotoxic concentrations (IC50 = 33 µg mL-1) on skin keratinocytes. In a mouse model of OA, the optimized formulation is able to attenuate inflammation and pain and minimize cartilage degeneration. Taken together, these data demonstrate the feasibility of adopting proniosome gels as a suitable platform to deliver active molecules for the management of osteoarthritis.

Investigations on detoxification mechanisms of novel para-phenylenediamine analogues through N-acetyltransferase 1 (NAT-1).

Para-phenylenediamine (PPD) is one of the most used chemicals in oxidative hair dyes. However, its use has been associated with adverse effects on health, including contact dermatitis and other systemic toxicities. Novel PPD derivatives have been proposed as a safer replacement for PPD. This can be achieved if these molecules minimally permeate the skin and/or are easily metabolised by enzymes in the skin (e.g., N-acetyltransferase-1 (NAT-1)) into innocuous compounds before gaining systemic entry. This study investigated the detoxification pathway mediated by NAT-1 enzymes on 6 synthesized PPD analogues (namely, P1-P6) with different chemical properties, to study the role of functional groups on detoxification mechanisms in HaCaT skin cells. These compounds were carefully designed with different chemical properties (whereby the ortho position of PPD was substituted by nucleophile and electrophile groups to promote N-acetylation reactions, metabolism and clearance). Compounds P2-P4 N-acetylated at 54-49 nmol/mg/min, which is 1.6 times higher than N-acetylation of PPD, upregulated NAT-1 activity from 8-7% at 50 µM to 22-11% at 100 µM and showed 4 times higher rate of elimination (k equal to 0.141 ± 0.016-0.124 ± 0.01 h-1) and 3 times faster rate of clearance (0.172 ± 0.007-0.158 ± 0.005 h-1mgprotein-1) than PPD (0.0316 ± 0.0019 h-1, 0.0576 ± 0.003 h-1mg protein-1, respectively). The data suggest that nucleophile substituted compounds detoxify at a faster rate than PPD. Our metabolic and detoxification mechanistic studies revealed significantly higher rates of N-acetylation, NAT-1 activity and higher detoxification of P2-P4 in keratinocytes, suggesting the importance of nucleophilic groups at the ortho position in PPD to reduce toxicity of aniline-based dyes on human skin cells.

Lyophilization Preserves the Intrinsic Cardioprotective Activity of Bioinspired Cell-Derived Nanovesicles.

Recently, bioinspired cell-derived nanovesicles (CDNs) have gained much interest in the field of nanomedicine due to the preservation of biomolecular structure characteristics derived from their parent cells, which impart CDNs with unique properties in terms of binding and uptake by target cells and intrinsic biological activities. Although the production of CDNs can be easily and reproducibly achieved with any kind of cell culture, application of CDNs for therapeutic purposes has been greatly hampered by their physical and chemical instability during long-term storage in aqueous dispersion. In the present study, we conceived a lyophilization approach that would preserve critical characteristics regarding stability (vesicles' size and protein content), structural integrity, and biological activity of CDNs for enabling long-term storage in freeze-dried form. Compared to the lyoprotectant sucrose, trehalose-lyoprotected CDNs showed significantly higher glass transition temperature and lower residual moisture content. As assessed by ATR-FTIR and far-UV circular dichroism, lyophilization in the presence of the lyoprotectant effectively maintained the secondary structure of cellular proteins. After reconstitution, lyoprotected CDNs were efficiently associated with HeLa cells, CT26 cells, and bone marrow-derived macrophages at a rate comparable to the freshly prepared CDNs. In vivo, both lyoprotected and freshly prepared CDNs, for the first time ever reported, targeted the injured heart, and exerted intrinsic cardioprotective effects within 24 h, attributable to the antioxidant capacity of CDNs in a myocardial ischemia/reperfusion injury animal model. Taken together, these results pave the way for further development of CDNs as cell-based therapeutics stabilized by lyophilization that enabled long-term storage while preserving their activity.

An exploratory analysis of effects of poyendarone, a deuterated analogue of dronedarone, on the canine model of paroxysmal atrial fibrillation.

Poyendarone, a deuterated analogue of dronedarone, is expected to reduce the onset of cardiovascular adverse events of dronedarone, including congestive heart failure and excessive QT-interval prolongation. Since information is still lacking on the anti-atrial fibrillatory property of poyendarone, we assessed it along with effects on the inter-atrial conduction time (IACT) and atrial effective refractory period (AERP) using the canine paroxysmal atrial fibrillation model. Poyendarone hydrochloride (n = 4) and dronedarone hydrochloride (n = 4) in intravenous doses of 0.3 and 3 mg/kg/30 s were cumulatively administered. Poyendarone hardly altered sinoatrial rate, but dronedarone decreased it in a dose-related manner, whereas both drugs slightly but significantly reduced idioventricular rate. Poyendarone shortened duration of burst pacing-induced atrial fibrillation, whereas such abbreviation was not observed by dronedarone. Poyendarone and dronedarone similarly prolonged IACT in a frequency-dependent manner, indicating that their INa inhibitory actions may be similar. The high dose of poyendarone prolonged AERP in a reverse frequency-dependent manner, extent of which at basic pacing cycle lengths of 300 and 400 ms was comparable to that of dronedarone. However, the extent at a basic pacing cycle length of 200 ms was tended to be greater in poyendarone than in dronedarone, suggesting greater IKs inhibitory action of poyendarone. The deuteration of dronedarone attenuated the inhibition of sinus automaticity and prolonged the AERP with keeping the blood pressure and ventricular rate stable. Thus, poyendarone may have both more potent anti-atrial fibrillatory action and wider cardiovascular safety margin than dronedarone.

Development of novel alternative hair dyes to hazardous para-phenylenediamine.

Most of the permanent hair dye products contain p-phenylenediamine (PPD), a well-known skin sensitizer. PPD may cause cutaneous reactions and leads to allergic contact dermatitis (ACD), a condition with major medical and financial repercussions. Hair dye-induced ACD represents a growing concern both for consumers and the cosmetics industry. In this study we introduced novel side chains on the PPD molecule with the goal of overcoming the hazard potential of PPD. Our strategy relies on the replacement of the colorless PPD with new, larger and intrinsically colorled PPD derivatives to reduce dermal penetration and thus the skin sensitization potential. We synthesized two oligomers with bulky side-chains, which displayed 7-8 times lower cytotoxicity than PPD, a significantly weaker sensitization potential (22.0 % and 23.8 % versus 55.5 % for PPD) in the Direct Peptide Reactivity Assay, minimal cumulative penetration through excised skin and an intrinsic ability to colour and preserve the nuance when applied on bleached hair. The lower skin permeation and sensitizing potential are absolutely crucial and give a clear advantage of our products over other standards. These novel PPD hair dyes show significantly less hazard potential than PPD and may, upon further risk assessment studies, replace PPD in consumer care products.

Micro cell vesicle technology (mCVT): a novel hybrid system of gene delivery for hard-to-transfect (HTT) cells.

A hybrid gene delivery platform, micro Cell Vesicle Technology (mCVT), produced from the fusion of plasma membranes and cationic lipids, is presently used to improve the transfection efficiency of hard-to-transfect (HTT) cells. The plasma membrane components of mCVTs impart specificity in cellular uptake and reduce cytotoxicity in the transfection process, while the cationic lipids complex with the genetic material and provide structural integrity to mCVTs.

Heteroleptic Ruthenium(II) Complexes with Bathophenanthroline and Bathophenanthroline Disulfonate Disodium Salt as Fluorescent Dyes for In-Gel Protein Staining.

The in-gel detection of proteins for various proteomic experiments is commonly done with the fluorescent RuII tris(bathophenanthroline disulfonate) complex (Ru(BPS)3), which is more cost-effective compared to commercial Ru-based formulations but requires tedious procedures for its preparation and strongly acidic staining conditions. Herein, we report the synthesis and characterization of heteroleptic RuII complexes Ru(BPS)2(BP) and Ru(BPS)(BP)2 containing bathophenanthroline (BP) and bathophenanthroline disulfonate disodium salt (BPS) in comparison with Ru(BPS)3. It was shown by fluorescent and UV-vis measurements that novel RuII complexes were excitable in both UV and visible light, close to emission bands of classical lasers, which is important for successful in-gel protein detection. Novel fluorescent dyes demonstrated improved protein detection in comparison with commercially available SYPRO Ruby staining solution. In addition, unlike commonly used staining protocols, staining with Ru(BPS)(BP)2 can be performed at nearly neutral pH, thereby reducing artificial post-translational modifications (PTMs).

How the Deuteration of Dronedarone Can Modify Its Cardiovascular Profile: In Vivo Characterization of Electropharmacological Effects of Poyendarone, a Deuterated Analogue of Dronedarone.

Since deuterium replacement has a potential to modulate pharmacodynamics, pharmacokinetics and toxicity, we developed deuterated dronedarone; poyendarone, and assessed its cardiovascular effects. Poyendarone hydrochloride in doses of 0.3 and 3 mg/kg over 30 s was intravenously administered to the halothane-anesthetized dogs (n = 4), which provided peak plasma concentrations of 108 ± 10 and 1120 ± 285 ng/mL, respectively. The 0.3 mg/kg shortened the ventricular repolarization period. The 3 mg/kg transiently increased the heart rate at 5 min but decreased at 45 min, and elevated the total peripheral vascular resistance and left ventricular preload, whereas it reduced the mean blood pressure at 5 min, left ventricular contractility and cardiac output. The transient tachycardic action is considered to be induced by the hypotension-induced, reflex-mediated increase of sympathetic tone. The 3 mg/kg delayed both intra-atrial and intra-ventricular conductions, indicating Na+ channel inhibitory action. Moreover, the 3 mg/kg transiently shortened the ventricular repolarization period at 5 min. No significant change was detected in the late repolarization by poyendarone, indicating it might not hardly significantly alter rapidly activating delayed-rectifier K+ current (IKr). Poyendarone prolonged the atrial effective refractory period greater than the ventricular parameter. When compared with dronedarone, poyendarone showed similar pharmacokinetics of dronedarone, but reduced ß-adrenoceptor blocking activity as well as the cardio-suppressive effect. Poyendarone failed to inhibit IKr and showed higher atrial selectivity in prolonging the effective refractory period of atrium versus ventricle. Thus, the deuteration may be an effective way to improve the cardiovascular profile of dronedarone. Poyendarone is a promising anti-atrial fibrillatory drug candidate.

Design, Synthesis and Evaluation of New Indolylpyrimidylpiperazines for Gastrointestinal Cancer Therapy.

Human A3 adenosine receptor hA3AR has been implicated in gastrointestinal cancer, where its cellular expression has been found increased, thus suggesting its potential as a molecular target for novel anticancer compounds. Observation made in our previous work indicated the importance of the carbonyl group of amide in the indolylpyrimidylpiperazine (IPP) for its human A2A adenosine receptor (hA2AAR) subtype binding selectivity over the other AR subtypes. Taking this observation into account, we structurally modified an indolylpyrimidylpiperazine (IPP) scaffold, 1 (a non-selective adenosine receptors' ligand) into a modified IPP (mIPP) scaffold by switching the position of the carbonyl group, resulting in the formation of both ketone and tertiary amine groups in the new scaffold. Results showed that such modification diminished the A2A activity and instead conferred hA3AR agonistic activity. Among the new mIPP derivatives (3-6), compound 4 showed potential as a hA3AR partial agonist, with an Emax of 30% and EC50 of 2.89 ± 0.55 µM. In the cytotoxicity assays, compound 4 also exhibited higher cytotoxicity against both colorectal and liver cancer cells as compared to normal cells. Overall, this new series of compounds provide a promising starting point for further development of potent and selective hA3AR partial agonists for the treatment of gastrointestinal cancers.

Discovery of indolylpiperazinylpyrimidines with dual-target profiles at adenosine A2A and dopamine D2 receptors for Parkinson's disease treatment.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra of the human brain, leading to depletion of dopamine production. Dopamine replacement therapy remains the mainstay for attenuation of PD symptoms. Nonetheless, the potential benefit of current pharmacotherapies is mostly limited by adverse side effects, such as drug-induced dyskinesia, motor fluctuations and psychosis. Non-dopaminergic receptors, such as human A2A adenosine receptors, have emerged as important therapeutic targets in potentiating therapeutic effects and reducing the unwanted side effects. In this study, new chemical entities targeting both human A2A adenosine receptor and dopamine D2 receptor were designed and evaluated. Two computational methods, namely support vector machine (SVM) models and Tanimoto similarity-based clustering analysis, were integrated for the identification of compounds containing indole-piperazine-pyrimidine (IPP) scaffold. Subsequent synthesis and testing resulted in compounds 5 and 6, which acted as human A2A adenosine receptor binders in the radioligand competition assay (Ki = 8.7-11.2 µM) as well as human dopamine D2 receptor binders in the artificial cell membrane assay (EC50 = 22.5-40.2 µM). Moreover, compound 5 showed improvement in movement and mitigation of the loss of dopaminergic neurons in Drosophila models of PD. Furthermore, in vitro toxicity studies on compounds 5 and 6 did not reveal any mutagenicity (up to 100 µM), hepatotoxicity (up to 30 µM) or cardiotoxicity (up to 30 µM).

Application of Static Modeling --in the Prediction of In Vivo Drug-Drug Interactions between Rivaroxaban and Antiarrhythmic Agents Based on In Vitro Inhibition Studies.

Rivaroxaban, a direct Factor Xa inhibitor, is indicated for stroke prevention in nonvalvular atrial fibrillation (AF). Studies have revealed that the clearance of rivaroxaban is largely attributed to CYP3A4, CYP2J2 metabolism, and P-glycoprotein (P-gp) efflux pathways. Amiodarone and dronedarone are antiarrhythmic agents employed in AF management. Amiodarone, dronedarone, and their major metabolites, N-desethylamiodarone (NDEA) and N-desbutyldronedarone (NDBD), demonstrate inhibitory effects on CYP3A4 and CYP2J2 with U.S. Food and Drug Administration-recommended probe substrates. In addition, both amiodarone and dronedarone are known P-gp inhibitors. Hence, the concomitant administration of these antiarrhythmic agents has the potential to augment the systemic exposure of rivaroxaban through simultaneous impairment of its clearance pathways. Currently, however, clinical data on the extent of these postulated drug-drug interactions are lacking. In this study, in vitro inhibition assays using rivaroxaban as the probe substrate demonstrated that both dronedarone and NDBD produced reversible inhibition as well as irreversible mechanism-based inactivation of CYP3A4- and CYP2J2-mediated metabolism of rivaroxaban. However, amiodarone and NDEA were observed to cause reversible inhibition as well as mechanism-based inactivation of CYP3A4 but not CYP2J2. In addition, amiodarone, NDEA, and dronedarone, but not NDBD, were determined to inhibit P-gp-mediated rivaroxaban transport. The in vitro inhibition parameters were fitted into a mechanistic static model, which predicted a 37% and 31% increase in rivaroxaban exposure due to the inhibition of hepatic and gut metabolism by amiodarone and dronedarone, respectively. A separate model quantifying the inhibition of P-gp-mediated efflux by amiodarone or dronedarone projected a 9% increase in rivaroxaban exposure.

Multiple modes of inhibition of human cytochrome P450 2J2 by dronedarone, amiodarone and their active metabolites.

Dronedarone, a multiple ion channel blocker is prescribed for the treatment of paroxysmal and persistent atrial fibrillation. While dronedarone does not precipitate toxicities like its predecessor amiodarone, its clinical use has been associated with idiosyncratic hepatic and cardiac adverse effects and drug-drug interactions (DDIs). As dronedarone is a potent mechanism-based inactivator of CYP3A4 and CYP3A5, a question arose if it exerts a similar inhibitory effect on CYP2J2, a prominent cardiac CYP450 enzyme. In this study, we demonstrated that CYP2J2 is reversibly inhibited by dronedarone (Ki=0.034 µM), amiodarone (Ki=4.8µM) and their respective pharmacologically active metabolites namely N-desbutyldronedarone (NDBD) (Ki=0.55 µM) and N-desethylamiodarone (NDEA) (Ki=7.4 µM). Moreover, time-, concentration- and NADPH-dependent irreversible inactivation of CYP2J2 was investigated where inactivation kinetic parameters (KI, kinact) and partition ratio (r) of dronedarone (0.05 µM, 0.034 min(-1), 3.3), amiodarone (0.21 µM, 0.015 min(-1), 20.7) and NDBD (0.48 µM, 0.024 min(-1), 21.7) were observed except for NDEA. The absence of the characteristic Soret peak, lack of recovery of CYP2J2 activity upon dialysis, and biotransformation of dronedarone and NDBD to quinone-oxime reactive metabolites further confirmed the irreversible inactivation of CYP2J2 by dronedarone and NDBD is via the covalent adduction of CYP2J2. Our novel findings illuminate the possible mechanisms of DDIs and cardiac adverse effects due to both reversible inhibition and irreversible inactivation of CYP2J2 by dronedarone, amiodarone and their active metabolites.

Plasma Metabonomic Profiling of Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and the leading cause of visual impairment in working-age adults. Patients with diabetes often develop DR despite appropriate control of systemic risk factors, suggesting the involvement of other pathogenic factors. We hypothesize that the plasma metabolic signature of DR is distinct and resolvable from that of diabetes alone. A nested population-based case-control metabonomic study was first performed on 40 DR cases and 40 control subjects with diabetes using gas chromatography-mass spectrometry. Eleven metabolites were found to be correlated with DR, and the majority were robust when adjusted for metabolic risk factors and confounding kidney disease. The metabolite markers 2-deoxyribonic acid; 3,4-dihydroxybutyric acid; erythritol; gluconic acid; and ribose were validated in an independent sample set with 40 DR cases, 40 control subjects with diabetes, and 40 individuals without diabetes. DR cases and control subjects with diabetes were matched by HbA1c in the validation set. Activation of the pentose phosphate pathway was identified from the list of DR metabolite markers. The identification of novel metabolite markers for DR provides insights into potential new pathogenic pathways for this microvascular complication and holds translational value in DR risk stratification and the development of new therapeutic measures.

Inactivation of Human Cytochrome P450 3A4 and 3A5 by Dronedarone and N-Desbutyl Dronedarone.

Dronedarone is an antiarrhythmic agent approved in 2009 for the treatment of atrial fibrillation. An in-house preliminary study demonstrated that dronedarone inhibits cytochrome P450 (CYP) 3A4 and 3A5 in a time-dependent manner. This study aimed to investigate the inactivation of CYP450 by dronedarone. We demonstrated for the first time that both dronedarone and its main metabolite N-desbutyl dronedarone (NDBD) inactivate CYP3A4 and CYP3A5 in a time-, concentration-, and NADPH-dependent manner. For the inactivation of CYP3A4, the inactivator concentration at the half-maximum rate of inactivation and inactivation rate constant at an infinite inactivator concentration are 0.87 µM and 0.039 minute(-1), respectively, for dronedarone, and 6.24 µM and 0.099 minute(-1), respectively, for NDBD. For CYP3A5 inactivation, the inactivator concentration at the half-maximum rate of inactivation and inactivation rate constant at an infinite inactivator concentration are 2.19 µM and 0.0056 minute(-1) for dronedarone and 5.45 µM and 0.056 minute(-1) for NDBD. The partition ratios for the inactivation of CYP3A4 and CYP3A5 by dronedarone are 51.1 and 32.2, and the partition ratios for the inactivation of CYP3A4 and CYP3A5 by NDBD are 35.3 and 36.6. Testosterone protected both CYP3A4 and CYP3A5 from inactivation by dronedarone and NDBD. Although the presence of Soret peak confirmed the formation of a quasi-irreversible metabolite-intermediate complex between dronedarone/NDBD and CYP3A4/CYP3A5, partial recovery of enzyme activity by potassium ferricyanide illuminated an alternative irreversible mechanism-based inactivation (MBI). MBI of CYP3A4 and CYP3A5 was further supported by the discovery of glutathione adducts derived from the quinone oxime intermediates of dronedarone and NDBD. In conclusion, dronedarone and NDBD inactivate CYP3A4 and CYP3A5 via unique dual mechanisms of MBI and formation of the metabolite-intermediate complex. Our novel findings contribute new knowledge for future investigation of the underlying mechanisms associated with dronedarone-induced hepatotoxicity and clinical drug-drug interactions.

Discovery of simplified N²-substituted pyrazolo[3,4-d]pyrimidine derivatives as novel adenosine receptor antagonists: efficient synthetic approaches, biological evaluations and molecular docking studies.

In the present study, a molecular simplification approach was employed to design novel bicyclic pyrazolo[3,4-d]pyrimidine (PP) derivatives from tricyclic pyrazolo[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines (PTP) as promising human A3 adenosine receptor (hA3AR) antagonists. All the target compounds were synthesized using novel and efficient synthetic schemes and the structure-activity relationship studies of these PPs were explored through the synthesis of a series of PTP analogues with various substituents. Substituents with different lipophilicity and steric hindrance (e.g., alkyl and aryl-alkyl) functions were introduced at N(2) position of the pyrazole ring, while acyl groups with different electronic properties were introduced at C(6) position of the bicyclic nucleus to probe both electronic and positional effects. Most of the synthesized derivatives of the PP series presented good affinity at the hA3AR, as indicated by the low micromolar range of Ki values and among them, compound 63 with N(2) neopentyl substituents showed most potent hA3AR affinity with Ki value of 0.9 µM and high selectivity (hA1AR/hA3AR=>111 & hA2AAR/hA3AR=>111) towards other adenosine receptor subtypes. Interestingly, small isopropyl groups at N(2) position displayed high affinity at another receptor subtype (hA2AAR, e.g., compound 55, with Ki hA2AAR=0.8 µM), while they were less favorable at the hA3AR. Molecular docking analysis was also performed to predict the possible binding mode of target compounds inside the hA3AR and hA2AAR. Overall, PP derivatives represent promising starting points for new AR antagonists.

Organoruthenium antagonists of human A3 adenosine receptors.

Human A3 adenosine receptor (A3AR) is a membrane-bound G protein-coupled receptor implicated in a number of severe pathological conditions, including cancer, in which it acts as a potential therapeutic target. To derive structure-activity relationships on pyrazolo-triazolo-pyrimidine (PTP)-based A3AR antagonists, we developed a new class of organometallic inhibitors through replacement of the triazolo moiety with an organoruthenium fragment. The objective was to introduce by design structural diversity into the PTP scaffold in order to tune their binding efficacy toward the target receptor. These novel organoruthenium antagonists displayed good aquatic stability and moderate binding affinity toward the hA3 receptor in the low micromolar range. The assembly of these complexes through a template-driven approach with selective ligand replacement at the metal center to control their steric and receptor-binding properties is discussed.