A Molecular Dynamic Simulation, Structural Analysis, and Ex Vivo Insights into the P-glycoprotein-Mediated Interactions of Dietary Polyphenols with Cyclin-dependent Kinase Inhibitors: A Potential Strategy to Counteract Drug Efflux.

INTRODUCTION: P-glycoprotein, an ATP-dependent efflux transporter, plays a crucial role in eliminating cellular toxins and affects the intracellular concentration and bioavailability of CDK 4/6 inhibitors. Moreover, dietary flavonoids are natural bio-enhancers that can effectively inhibit the efflux function of these transporters. Therefore, this study aimed to assess the impact of dietary polyphenols on the inhibition of P-glycoprotein and the subsequent efflux of CDK inhibitors palbociclib and ribociclib. METHODS: A molecular docking approach was implemented to evaluate the binding interaction characteristics of CDK4/6 inhibitors in the presence of dietary polyphenols at the ATP binding site. Furthermore, the stability of the complexes was evaluated in two conformations of P-glycoprotein, followed by an ex vivo everted gut sac experiment. RESULTS: The findings demonstrated that the binding of curcumin and quercetin with high affinity (-51.63 and 47.16 Kcal/mol) to ATP binding sites of P-glycoprotein-palbociclib and ribociclib inward conformation complexes resulted in good stability of complex and minimal fluctuation throughout the course of the simulation. It was evident from the everted gut sac ex vivo study that the presence of 100µM of curcumin resulted in an increase of 1.77 and 4.20-fold in the intestinal transit of palbociclib and ribociclib, respectively. CONCLUSION: The study emphasizes the significance of curcumin and quercetin as inhibitors of P-glycoprotein, demonstrating their potential to decrease the efflux of palbociclib and ribociclib, consequently contributing to their bioavailability enhancement.

Targeting next-generation PDE4 inhibitors in search of potential management of rheumatoid arthritis and psoriasis.

Immune-mediated inflammatory diseases (IMIDs) comprise a broad spectrum of conditions characterized by systemic inflammation affecting various organs and tissues, for which there is no known cure. The isoform-specific inhibition of phosphodiesterase-4B (PDE4B) over PDE4D constitutes an effective therapeutic strategy for the treatment of IMIDs that minimizes the adverse effects associated with non-selective PDE4 inhibitors. Thus, we report a new class of isoquinolone derivatives as next-generation PDE4 inhibitors for effective management of rheumatoid arthritis (RA) and psoriasis. Among the series, 8 compounds i.e. 1e, 1l, 1m, 1n, 1o, 2m, 2o and 3o showed promising PDE4B inhibition (>80 %) in vitro with IC50 ∼ 1.4-6.2 µM. The compound 1l was identified as an initial hit and was pursued for further studies. According to structure-activity relationship (SAR), an allyl group at C-4 position improved PDE4B inhibition. The correlation between in vitro activity data and binding affinities obtained via molecular docking suggested that the high-affinity binding to PDE4B is a prerequisite for the effective inhibition of PDE4B. Notably, the hit 1l showed selectivity towards PDE4B over PDE4D in vitro. Furthermore, 1l treatment (30 mg/kg) in the adjuvant-induced arthritis (AIA) rat model induced by complete Freund's adjuvant (CFA) demonstrated anti-arthritic potential via ameliorating paw swelling and body weight, narrowing joint space, reducing excessive immune cells infiltration and pannus formation in addition to reducing mRNA expression of pro-inflammatory cytokines such as TNF-α and IL-6 in synovial tissues of experimental rats. Additionally, 1l reduced the hyper-proliferative state and colony forming potential of IMQ-induced psoriatic keratinocytes. The treatment of these cells with 1l markedly reduced the protein levels of Ki67 and mRNA levels of pro-inflammatory cytokines e.g. IL-17A and TNF-α suggesting its potent anti-psoriatic potential. Furthermore, 1l did not show any significant adverse effects when evaluated in a systematic toxicity (e.g. teratogenicity, hepatotoxicity and cardiotoxicity) studies in zebrafish at the tested concentrations (1-100 µM) and the NOAEL (no-observed-adverse-effect level) was found to be 100 µM. Thus, with promising anti-inflammatory effects both in vitro and in vivo along with PDE4B selectivity with an acceptable safety margin, 1l emerged as a new and promising inhibitor for further studies.

Design, synthesis and evaluation of 2-aryl quinoline derivatives against 12R-lipoxygenase (12R-LOX): Discovery of first inhibitor of 12R-LOX.

The 12R-lipoxygenase (12R-LOX), a (non-heme) iron-containing metalloenzyme belonging to the lipoxygenase (LOX) family catalyzes the conversion of arachidonic acid (AA) to its key metabolites. Studies suggested that 12R-LOX plays a critical role in immune modulation for the maintenance of skin homeostasis and therefore can be considered as a potential drug target for psoriasis and other skin related inflammatory diseases. However, unlike 12-LOX (or 12S-LOX) the enzyme 12R-LOX did not receive much attention till date. In our effort, the 2-aryl quinoline derivatives were designed, synthesized and evaluated for the identification of potential inhibitors of 12R-hLOX. The merit of selection of 2-aryl quinolines was assessed by in silico docking studies of a representative compound (4a) using the homology model of 12R-LOX. Indeed, in addition to participating in H-bonding with THR628 and LEU635 the molecule formed a hydrophobic interaction with VAL631. The desired 2-aryl quinolines were synthesized either via the Claisen-Schmidt condensation followed by one-pot reduction-cyclization or via the AlCl3 induced heteroarylation or via the O-alkylation approach in good to high (82-95%) yield. When screened against human 12R-LOX (12R-hLOX) in vitro four compounds (e.g. 4a, 4d, 4e and 7b) showed encouraging (>45%) inhibition at 100 µM among which 7b and 4a emerged as the initial hits. Both the compounds showed selectivity towards 12R-hLOX over 12S-hLOX, 15-hLOX and 15-hLOXB and concentration dependent inhibition of 12R-hLOX with IC50 = 12.48 ± 2.06 and 28.25 ± 1.63 µM, respectively. The selectivity of 4a and 7b towards 12R-LOX over 12S-LOX was rationalized with the help of molecular dynamics simulations. The SAR (Structure-Activity Relationship) within the present series of compounds suggested the need of a o-hydroxyl group on the C-2 phenyl ring for the activity. The compound 4a and 7b (at 10 and 20 µM) reduced the hyper-proliferative state and colony forming potential of IMQ-induced psoriatic keratinocytes in a concentration dependent manner. Further, both compounds decreased the protein levels of Ki67 and the mRNA expression of IL-17A in the IMQ-induced psoriatic-like keratinocytes. Notably, 4a but not 7b inhibited the production of IL-6 and TNF-α in the keratinocyte cells. In the preliminary toxicity studies (i.e. teratogenicity, hepatotoxicity and heart rate assays) in zebrafish both the compounds showed low safety (<30 µM) margin. Overall, being the first identified inhibitors of 12R-LOX both 4a and 7b deserve further investigations.

Fe(III)-catalyzed regioselective and faster synthesis of isocoumarins with 3-oxoalkyl moiety at C-4: Identification of new inhibitors of PDE4.

In search of potent and new anti-inflammatory agents, we explored a new class of isocoumarin derivatives possessing the 3-oxoalkyl moiety at C-4 position. These compounds were synthesized via the FeCl3 catalyzed construction of isocoumarin ring. The methodology involved coupling of 2-alkynyl benzamides with alkyl vinyl ketone and proceeded via a regioselective cyclization to give the desired compound as a result of formation of CO and CC bonds. A large number of isocoumarins were synthesized and assessed against PDE4B in vitro. While isocoumarins containing an aminosulfonyl moiety attached to the C-3 aryl ring showed encouraging inhibition of PDE4B, some of the derivatives devoid of aminosulfonyl moiety also showed considerable inhibition. According to the SAR analysis the C6H4NHSO2R2-m moiety at C-3 position of the isocoumarin ring was favorable when the R2 was chosen as an aryl or 2-thienyl group whereas the presence of F or OMe substituent at C-7 of the isocoumarin ring was found to be beneficial. The compound 5f with IC50 values 0.125 ± 0.032 and 0.43 ± 0.013 µM against PDE4B and 4D, respectively was identified as an initial hit. It showed in silico interaction with the PHE678 residue in the CR3 region of PDE4B and relatively less number of interactions with PDE4D. Besides showing the PDE4 selectivity over other PDEs and TNF-α inhibition in vitro the compound 5f at an intraperitoneal dose of 30 mg/kg demonstrated the protective effects against the development of arthritis and potent immunomodulatory activity in adjuvant induced arthritic (AIA) rats. Furthermore, no significant adverse effects were observed for this compound when evaluated in a systematic toxicity (e.g. teratogenicity, hepatotoxicity and cardiotoxicity) studies in zebrafish at various concentrations. Collectively, being a new, potent, moderately selective and safe inhibitor of PDE4B the isocoumarin 5f can be progressed into further pharmacological studies.

Simultaneous Estimation of Apremilast and Betamethasone Dipropionate in Microsponge-Based Topical Formulation using a Stability Indicating RP-HPLC Method: A Quality-by-Design Approach.

A stability-indicating reverse phase high-performance liquid chromatography method was developed and validated for simultaneous quantification of apremilast (APL) and betamethasone dipropionate (BD) in bulk as well as drug loaded microsponges. Various mobile phase systems were screened to check the system suitability followed by force degradation analysis to determine APL and BD stability under varying stress conditions. A central composite design model was used to optimize the column temperature and flow rate using Design Expert® (9.0.1). One factor at a time approach with five independent factors were used to validate the robustness of the method. Finally, APL and BD were precisely and accurately quantified from drug loaded microsponges using the validated method. A favorable separation of APL and BD was obtained on a Phenomenex® Luna C18 column using a mixture of 50 mM phosphate buffer containing 0.1% triethylamine (pH 6.1) and acetonitrile (60:40%v/v) as mobile phase. Both the drugs were found to be stable when exposed to stressors such as heat-, light-, alkali-, acid- and peroxide-induced degradation. The calibration curves were found to be linear with appreciable limit of detection and limit of quantification. Recovery and percentage relative standard deviation of peak areas for APL and BD were found to be < 2.0% and 99-100% in bulk drug solution and <2.0% and 99-103% in microsponge formulation, respectively. Statistical analysis using analysis of variance indicated that the model was significant (P < 0.001). Hence, the developed method can be effectively used to quantify APL and BD, both in bulk as well as microsponge formulations.

Enhanced ex vivo intestinal absorption of olmesartan medoxomil nanosuspension: Preparation by combinative technology.

The purpose of this study was to develop nanosuspension based on combinative technology to enhance the intestinal absorption of Olmesartan medoxomil (OLM), a potent antihypertensive agent with limited oral bioavailability. Two combinative approaches were employed and then characterized. In vitro intestinal absorption of OLM nanosuspension and plain OLM was studied using non-everted rat intestinal sac model. Optimal OLM nanosuspension was prepared by a combination of ball milling and probe sonication using stabilizer, Poloxamer 407. The formula exhibited particle size of 469.9 nm and zeta potential of -19.1 mV, which was subjected to ex vivo studies. The flux and apparent permeability coefficient in intestine from OLM nanosuspension was higher than the plain drug, thereby suggesting better drug delivery.