Enantioselective polar-organic mode high-performance liquid chromatographic separation of lifitegrast on immobilized polysaccharide stationary phase and its application to pH-dependent chiral interconversion studies.

(S)-Lifitegrast (LFT) is the novel integrin antagonist, approved by the Food and drug administration, to treat signs and symptoms of dry eye disease. Synthesis of racemic LFT, preparative and analytical enantiomer separation, and chiral interconversion studies are lacking in the literature. Hence, in our study, synthesis of LFT racemate, chiral preparative purification procedure of enantiomer, and comprehensive analytical advancements are focused on rapid enantioselective separation and pH-dependent chiral interconversion studies. The synthesis of LFT racemate employed 2-amino-3-(3-(methylsulfonyl)phenyl)propanoic acid hydrochloride and 2-(benzofuran-6-carbonyl)-5,7-dichloro-1,2,3,4-tetrahydroisoquinoline-6-carbonyl chloride as starting materials. (R)-LFT was isolated from the racemate by preparative chiral HPLC and characterized using Q-TOF, FT-IR, NMR spectroscopy, and chiral HPLC. The purity of (R)-LFT was determined to have an enantiomeric excess of 99.12%. A precise, accurate, rapid HPLC-DAD enantioselective analytical method has been developed on Chiralpak IC [tris(3,5-dichloro phenyl carbamate) immobilized on cellulose] using water and methanol as mobile phase. The chiral interconversion study reveals 0.22% and 0.21% of interconversion of (S)-LFT into (R)-LFT at 80°C in pH 7.4 and 9.5 buffers, respectively, on the 24th day. An alternative route to enantioselective synthesis of LFT enantiomers by chromatographic separation is proposed. The validated enantioselective HPLC method will help to test the regular quality control samples.

Phytochemically analysed extract of Ageratina adenophora (Sprengel) R.M.King & H. Rob. initiates caspase 3-dependant apoptosis in colorectal cancer cell: A synergistic approach with chemotherapeutic drugs.

ETHNOPHARMACOLOGICAL RELEVANCE: Ageratina adenophora (Sprengel) R.M.King & H.Rob. has been used as traditional indigenous medicine all across the globe for its diverse therapeutic applications such as anticancer, analgesic, antipyretic, thermogenic, antiseptic, antimicrobial as well as astringent. The various ethnic groups of India use plant parts to treat cuts and wounds, venomous insect bites, skin lesions, blisters, scabies and other skin irritations, gastritis and indigestion problems, cough, stomach ache and dysentery. The Portuguese traditionally extract the juice from the plant and use it for cancer, diabetes, liver disorder, gallbladder and stomach ailments. Nigerian healers use different parts of the plant to treat diabetes, fever and inflammation. AIM OF THE STUDY: The aim of this study is to investigate the cytotoxic potential of A. adenophora hydroalcoholic leaves extract (AHL) on Colorectal cancer (CRC) cell lines (HCT-116, HCT-15 and HT-29), synergistic potential with chemotherapeutic drugs 5FU and Cisplatin as well as reactive oxygen species (ROS) generation, based on the sample collected from Mao district of Manipur, India. Identification of bioactive phytocompounds in AHL was also performed by HRLCMS. METHODS: The AHL was evaluated for its cytotoxic as well as antiproliferative activities by 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay, clonogenic and cell migration assays. The total phenolic content (TPC) and total flavonoid content (TFC) were quantified by Folin-ciocalteu and Aluminium chloride assays respectively. Caspase 3 activation was evaluated using Caspase-3 Assay Kit. Apoptosis detection by flow cytometry was carried out using annexin V-FITC/PI apoptosis detection kit. The apoptotic cells were also visualized by Giemsa and 4',6-Diamidino-2-phenylindole (DAPI) staining. The intracellular Reactive oxygen species (ROS) generation was also evaluated using fluorescent probe 2',7'-dichlorodihydrofluorescein di-acetate (H2DCFDA) in flow cytometry. The combination effects of AHL with chemotherapeutic drugs 5FU and Cisplatin were also evaluated. The identification of phytochemical constituents of AHL were analysed by HR-LCMS. RESULTS: The AHL induced cytotoxic activity significantly in HCT-116 with IC50 of 65.65 ± 2.10 µg/mL, but non-cancerous cell HeK-293 was least cytotoxic. Colony formation and cell migration were inhibited in a dose and time dependent manner. The cell morphology upon AHL treatment was significantly altered with apoptotic features. The extract was rich in total phenolic (82.09 ± 0.35mgGAE/g) and total flavonoid (58.31 ± 0.55 mgQAE/g) contents. AHL induced apoptosis as detected by AnnexinV/PI, via activation of caspase 3 and elevated production of Reactive oxygen species (ROS). AHL in combination with 5FU and Cisplatin acts synergistically and potentiates the therapeutic properties of the extract. Sesquiterpenes, phenolic as well as flavonoid derivatives with anticancer properties were detected in AHL by HRLCMS, and these phytoconstituents may be attributed for anticancer property of AHL. CONCLUSION: The present study evaluates the effectiveness of AHL against Colorectal cancer cell lines. AHL is cytotoxic and induces apoptosis in HCT-116 cells by caspase 3 activation and increased ROS production that can be attributed to sesquiterpenoids. Thus, the plant A. adenophora has therapeutic potential for Colorectal cancer and can be further exploited for developing anticancer drug.

Development of a Minimalistic Physiologically Based Pharmacokinetic (mPBPK) Model for the Preclinical Development of Spectinamide Antibiotics.

Spectinamides 1599 and 1810 are lead spectinamide compounds currently under preclinical development to treat multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. These compounds have previously been tested at various combinations of dose level, dosing frequency, and route of administration in mouse models of Mycobacterium tuberculosis (Mtb) infection and in healthy animals. Physiologically based pharmacokinetic (PBPK) modeling allows the prediction of the pharmacokinetics of candidate drugs in organs/tissues of interest and extrapolation of their disposition across different species. Here, we have built, qualified, and refined a minimalistic PBPK model that can describe and predict the pharmacokinetics of spectinamides in various tissues, especially those relevant to Mtb infection. The model was expanded and qualified for multiple dose levels, dosing regimens, routes of administration, and various species. The model predictions in mice (healthy and infected) and rats were in reasonable agreement with experimental data, and all predicted AUCs in plasma and tissues met the two-fold acceptance criteria relative to observations. To further explore the distribution of spectinamide 1599 within granuloma substructures as encountered in tuberculosis, we utilized the Simcyp granuloma model combined with model predictions in our PBPK model. Simulation results suggest substantial exposure in all lesion substructures, with particularly high exposure in the rim area and macrophages. The developed model may be leveraged as an effective tool in identifying optimal dose levels and dosing regimens of spectinamides for further preclinical and clinical development.

Efficacy of a brain-penetrant antiviral in lethal Venezuelan and eastern equine encephalitis mouse models.

Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV, respectively) are mosquito-borne, neuroinvasive human pathogens for which no FDA-approved therapeutic exists. Besides the biothreat posed by these viruses when aerosolized, arthropod transmission presents serious health risks to humans, as demonstrated by the 2019 outbreak of EEE disease in the United States that resulted in 38 confirmed cases, 19 deaths, and neurological effects in survivors. Here, we describe the discovery of a 2-pyrrolidinoquinazolinone scaffold, efficiently synthesized in two to five steps, whose structural optimization resulted in profound antiviral activity. The lead quinazolinone, BDGR-49, potently reduced cellular VEEV and EEEV titers by >7 log at 1 µM and exhibited suitable intravenous and oral pharmacokinetic profiles in BALB/c mice to achieve excellent brain exposure. Outstanding in vivo efficacy was observed in several lethal, subcutaneous infection mouse models using an 8-day dosing regimen. Prophylactically administered BDGR-49 at 25 mg kg-1 per day fully protected against a 10× LD50 VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD50 EEEV FL93-939-infected C57BL/6 mice were treated prophylactically with BDGR-49 at 50 mg kg-1 per day. Last, we observed 100% therapeutic efficacy when mice, challenged with 10× LD50 VEEV TrD, were dosed at 48 hours after infection with BDGR-49 at 25 mg kg-1 per day. Mouse brain viral titers at 96 hours after infection were reduced to values near the limit of detection. Collectively, these results underscore the substantial development potential of a well-tolerated, brain-penetrant lead compound that shows promise in preventing and treating encephalitic alphavirus disease.

Novel, Brain-Permeable, Cross-Species Benzothiazole Inhibitors of Microsomal Prostaglandin E Synthase-1 (mPGES-1) Dampen Neuroinflammation In Vitro and In Vivo.

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme of the cyclooxygenase (COX) cascade that generates prostaglandin E2 (PGE2) during inflammatory conditions. PGE2 is known to be a potent immune signaling molecule that mediates both peripheral and central inflammations. Inhibition of mPGES-1, rather than COX, may overcome the cardiovascular side effects associated with long-term COX inhibition by providing a more specific strategy to target inflammation. However, mPGES-1 inhibitor development is hampered by the large differences in cross-species activity due to the structural differences between the human and murine mPGES-1. Here, we report that our thiazole-based mPGES-1 inhibitors, compounds 11 (UT-11) and 19 derived from two novel scaffolds, were able to suppress PGE2 production in human (SK-N-AS) and murine (BV2) cells. The IC50 values of inhibiting PGE2 production in human and murine cells were 0.10 and 2.00 µM for UT-11 and 0.43 and 1.55 µM for compound 19, respectively. Based on in vitro and in vivo pharmacokinetic data, we selected UT-11 for evaluation in a lipopolysaccharide (LPS)-induced inflammation model. We found that our compound significantly suppressed proinflammatory cytokines and chemokines in the hippocampus but not in the kidney. Taken together, we demonstrated the potential of UT-11 in treating neuroinflammatory conditions, including epilepsy and stroke, and warrant further optimization.

Design, Synthesis, and Biological Evaluation of Pyrimidine Dihydroquinoxalinone Derivatives as Tubulin Colchicine Site-Binding Agents That Displayed Potent Anticancer Activity Both In Vitro and In Vivo.

Polymerization of tubulin dimers to form microtubules is one of the key events in cell proliferation. The inhibition of this event has long been recognized as a potential treatment option for various types of cancer. Compound 1e was previously developed by our team as a potent inhibitor of tubulin polymerization that binds to the colchicine site. To further improve the potency and therapeutic properties of compound 1e, we hypothesized based on the X-ray crystal structure that modification of the pyrimidine dihydroquinoxalinone scaffold with additional hetero-atom (N, O, and S) substituents could allow the resulting new compounds to bind more tightly to the colchicine site and display greater efficacy in cancer therapy. We therefore synthesized a series of new pyrimidine dihydroquinoxalinone derivatives, compounds 10, 12b-c, 12e, 12h, and 12j-l, and evaluated their cytotoxicity and relative ability to inhibit proliferation, resulting in the discovery of new tubulin-polymerization inhibitors. Among these, the most potent new inhibitor was compound 12k, which exhibited high cytotoxic activity in vitro, a longer half-life than the parental compound in liver microsomes (IC50 = 0.2 nM, t 1/2 = >300 min), and significant potency against a wide range of cancer cell lines including those from melanoma and breast, pancreatic, and prostate cancers. High-resolution X-ray crystal structures of the best compounds in this scaffold series, 12e, 12j, and 12k, confirmed their direct binding to the colchicine site of tubulin and revealed their detailed molecular interactions. Further evaluation of 12k in vivo using a highly taxane-resistant prostate cancer xenograft model, PC-3/TxR, demonstrated the strong tumor growth inhibition at the low dose of 2.5 mg/kg (i.v., twice per week). Collectively, these results strongly support further preclinical evaluations of 12k as a potential candidate for development.

SB226, an inhibitor of tubulin polymerization, inhibits paclitaxel-resistant melanoma growth and spontaneous metastasis.

Extensive preclinical studies have shown that colchicine-binding site inhibitors (CBSIs) are promising drug candidates for cancer therapy. Although numerous CBSIs were generated and evaluated, but so far the FDA has not approved any of them due to undesired adverse events or insufficient efficacies. We previously reported two very potent CBSIs, the dihydroquinoxalinone compounds 5 m and 5t. In this study, we further optimized the structures of compounds 5 m and 5t and integrated them to generate a new analog, SB226. X-ray crystal structure studies and a tubulin polymerization assay confirmed that SB226 is a CBSI that could disrupt the microtubule dynamics and interfere with microtubule assembly. Biophysical measurements using surface plasmon resonance (SPR) spectroscopy verified the high binding affinity of SB226 to tubulin dimers. The in vitro studies showed that SB226 possessed sub-nanomolar anti-proliferative activities with an average IC50 of 0.76 nM against a panel of cancer cell lines, some of which are paclitaxel-resistant, including melanoma, breast cancer and prostate cancer cells. SB226 inhibited the colony formation and migration of Taxol-resistant A375/TxR cells, and induced their G2/M phase arrest and apoptosis. Our subsequent in vivo studies confirmed that 4 mg/kg SB226 strongly inhibited the tumor growth of A375/TxR melanoma xenografts in mice and induced necrosis, anti-angiogenesis, and apoptosis in tumors. Moreover, SB226 treatment significantly inhibited spontaneous axillary lymph node, lung, and liver metastases originating from subcutaneous tumors in mice without any obvious toxicity to the animals' major organs, demonstrating the therapeutic potential of SB226 as a novel anticancer agent for cancer therapy.

Intranasal delivery of darunavir improves brain drug concentrations in mice for effective HIV treatment.

Despite the availability of combined antiretroviral therapy (cART), which reduces the HIV replication in chronically HIV-infected patients, HIV associated neurocognitive disorders (HAND) persists in the brain. The blood-brain barrier (BBB) is the major barrier for the penetration of drugs including antiretrovirals, limiting the drug penetration to the brain. In the present study, we have shown improved brain drug concentration in mice for darunavir (DRV), an FDA-approved drug, using an intranasal (IN) delivery method that bypasses the BBB. Here, we compared the time-dependent biodistribution of DRV at two different concentrations, high (25 mg/kg) and low (2.5 mg/kg), using two administration routes intravenous (IV) and intranasal (IN) in brain, liver, lungs, and plasma. Compared with IV administration, IN administration demonstrated a significantly improved DRV penetration in the brain at both low and high DRV concentrations (IV vs IN: at 2.5 mg/kg: 6.91 ± 1.69 ng/g vs 12.08 ± 2.91 ng/g, at 25 mg/kg: 12.84 ± 2.88 ng/g vs 19.74 ± 1.80 ng/g). As expected, IN administration showed significantly lower DRV concentrations in plasma (IV vs IN: at 2.5 mg/kg: 81.37 ± 22.04 ng/g vs 19.91 ± 12.65 ng/g, at 25 mg/kg: 899.12 ± 136.93 ng/g vs 320.56 ± 40.04 ng/g) and liver (IV vs IN: at 2.5 mg/kg: 118.39 ± 28.13 ng/g vs 29.27 ± 4.17 ng/g at 25 mg/kg: 1085.18 ± 255.0 ng/g vs 833.83 ± 242.4 ng/g). The IN administration did not show significant change in lungs compared to the IV administration. As a result, these findings suggest that the IN route can increase the DRV level in the brain, suppressing HIV in the brain reservoirs. Additionally, it could also reduce off-target effects, especially in peripheral organs.

Preclinical Evaluation of Inhalational Spectinamide-1599 Therapy against Tuberculosis.

The lengthy treatment time for tuberculosis (TB) is a primary cause for the emergence of multidrug resistant tuberculosis (MDR-TB). One approach to improve TB therapy is to develop an inhalational TB therapy that when administered in combination with oral TB drugs eases and shortens treatment. Spectinamides are new semisynthetic analogues of spectinomycin with excellent activity against Mycobacterium tuberculosis (Mtb), including MDR and XDR Mtb strains. Spectinamide-1599 was chosen as a promising candidate for development of inhalational therapy. Using the murine TB model and intrapulmonary aerosol delivery of spectinamide-1599, we characterized the pharmacokinetics and efficacy of this therapy in BALB/c and C3HeB/FeJ mice infected with the Mtb Erdman strain. As expected, spectinamide-1599 exhibited dose-dependent exposure in plasma, lungs, and ELF, but exposure ratios between lung and plasma were 12-40 times higher for intrapulmonary compared to intravenous or subcutaneous administration. In chronically infected BALB/c mice, low doses (10 mg/kg) of spectinamide-1599 when administered thrice weekly for two months provide efficacy similar to that of higher doses (50-100 mg/kg) after one month of therapy. In the C3HeB/FeJ TB model, intrapulmonary aerosol delivery of spectinamide-1599 (50 mg/kg) or oral pyrazinamide (150 mg/kg) had limited or no efficacy in monotherapy, but when both drugs were given in combination, a synergistic effect with superior bacterial reduction of >1.8 log10 CFU was observed. Throughout the up to eight-week treatment period, intrapulmonary therapy was well-tolerated without any overt toxicity. Overall, these results strongly support the further development of intrapulmonary spectinamide-1599 as a combination partner for anti-TB therapy.

X-ray Crystallography-Guided Design, Antitumor Efficacy, and QSAR Analysis of Metabolically Stable Cyclopenta-Pyrimidinyl Dihydroquinoxalinone as a Potent Tubulin Polymerization Inhibitor.

Small molecules that interact with the colchicine binding site in tubulin have demonstrated therapeutic efficacy in treating cancers. We report the design, syntheses, and antitumor efficacies of new analogues of pyridopyrimidine and hydroquinoxalinone compounds with improved drug-like characteristics. Eight analogues, 5j, 5k, 5l, 5m, 5n, 5r, 5t, and 5u, showed significant improvement in metabolic stability and demonstrated strong antiproliferative potency in a panel of human cancer cell lines, including melanoma, lung cancer, and breast cancer. We report crystal structures of tubulin in complex with five representative compounds, 5j, 5k, 5l, 5m, and 5t, providing direct confirmation for their binding to the colchicine site in tubulin. A quantitative structure-activity relationship analysis of the synthesized analogues showed strong ability to predict potency. In vivo, 5m (4 mg/kg) and 5t (5 mg/kg) significantly inhibited tumor growth as well as melanoma spontaneous metastasis into the lung and liver against a highly paclitaxel-resistant A375/TxR xenograft model.

Model-Based Exposure-Response Assessment for Spectinamide 1810 in a Mouse Model of Tuberculosis.

Despite decades of research, tuberculosis remains a leading cause of death from a single infectious agent. Spectinamides are a promising novel class of antituberculosis agents, and the lead spectinamide 1810 has demonstrated excellent efficacy, safety, and drug-like properties in numerous in vitro and in vivo assessments in mouse models of tuberculosis. In the current dose ranging and dose fractionation study, we used 29 different combinations of dose level and dosing frequency to characterize the exposure-response relationship for spectinamide 1810 in a mouse model of Mycobacterium tuberculosis infection and in healthy animals. The obtained data on 1810 plasma concentrations and counts of CFU in lungs were analyzed using a population pharmacokinetic/pharmacodynamic (PK/PD) approach as well as classical anti-infective PK/PD indices. The analysis results indicate that there was no difference in the PK of 1810 in infected compared to healthy, uninfected animals. The PK/PD index analysis showed that bacterial killing of 1810 in mice was best predicted by the ratio of maximum free drug concentration to MIC (fCmax/MIC) and the ratio of the area under the free concentration-time curve to the MIC (fAUC/MIC) rather than the cumulative percentage of time that the free drug concentration is above the MIC (f%TMIC). A novel PK/PD model with consideration of postantibiotic effect could adequately describe the exposure-response relationship for 1810 and supports the notion that the in vitro observed postantibiotic effect of this spectinamide also translates to the in vivo situation in mice. The obtained results and pharmacometric model for the exposure-response relationship of 1810 provide a rational basis for dose selection in future efficacy studies of this compound against M. tuberculosis.

Detection of glutathione conjugates of amiodarone and its reactive diquinone metabolites in rat bile using mass spectrometry tools.

RATIONALE: Amiodarone is reported to cause hepato and pulmonary toxicity in humans, which has been envisaged to be due to formation of its reactive metabolites, essentially based on its structural similarity to benzbromarone, a drug withdrawn from the market due to reasons of similar hepatotoxicity. Therefore, the purpose of this study was to detect glutathione conjugates of amiodarone and its reactive diquinone metabolites in rat bile using mass spectrometry tools. METHODS: Wistar rats were dosed orally with an amiodarone suspension and bile was collected via bile duct cannulation followed by solid-phase extraction, protein precipitation and centrifugation. Samples were analysed by liquid chromatography coupled with linear ion trap mass spectrometry using tandem mass and constant neutral loss scan in positive electrospray ionization mode. RESULTS: Glutathione adducts of amiodarone and its reactive diquinone metabolites were identified and characterized with the characteristic neutral loss of 129 Da. Glucuronide conjugates of previously reported stable phase-1 metabolites were also observed. CONCLUSIONS: This study confirmed generation of reactive metabolites of amiodarone for the first time, as was hypothesised earlier by various research groups. Also, the responsible toxicophore was identified to be a benzofuran moiety liable to form reactive diquinone species. However, the results need to be further confirmed in human subjects. Copyright © 2016 John Wiley & Sons, Ltd.