Design, Synthesis, and Pharmacological Evaluation of Embelin-Aryl/alkyl Amine Hybrids as Orally Bioavailable Blood-Brain Barrier Permeable Multitargeted Agents with Therapeutic Potential in Alzheimer's Disease: Discovery of SB-1448.

The complex and multifaceted nature of Alzheimer's disease has brought about a pressing demand to develop ligands targeting multiple pathways to combat its outrageous prevalence. Embelin is a major secondary metabolite of Embelia ribes Burm f., one of the oldest herbs in Indian traditional medicine. It is a micromolar inhibitor of cholinesterases (ChEs) and ß-site amyloid precursor protein cleaving enzyme 1 (BACE-1) with poor absorption, distribution, metabolism, and excretion (ADME) properties. Herein, we synthesize a series of embelin-aryl/alkyl amine hybrids to improve its physicochemical properties and therapeutic potency against targeted enzymes. The most active derivative, 9j (SB-1448), inhibits human acetylcholinesterase (hAChE), human butyrylcholinesterase (hBChE), and human BACE-1 (hBACE-1) with IC50 values of 0.15, 1.6, and 0.6 µM, respectively. It inhibits both ChEs noncompetitively with ki values of 0.21 and 1.3 µM, respectively. It is orally bioavailable, crosses blood-brain barrier (BBB), inhibits Aß self-aggregation, possesses good ADME properties, and protects neuronal cells from scopolamine-induced cell death. The oral administration of 9j at 30 mg/kg attenuates the scopolamine-induced cognitive impairments in C57BL/6J mice.

In vitro and in vivo anticancer potential and molecular targets of the new colchicine analog IIIM-067.

OBJECTIVE: The current study evaluated various new colchicine analogs for their anticancer activity and to study the primary mechanism of apoptosis and in vivo antitumor activity of the analogs with selective anticancer properties and minimal toxicity to normal cells. METHODS: Sulforhodamine B (SRB) assay was used to screen various colchicine analogs for their in vitro cytotoxicity. The effect of N-[(7S)-1,2,3-trimethoxy-9-oxo-10-(pyrrolidine-1-yl)5,6,7,9-tetrahydrobenzo[a] heptalene-7-yl] acetamide (IIIM-067) on clonogenicity, apoptotic induction, and invasiveness of A549 cells was determined using a clonogenic assay, scratch assay, and staining with 4',6-diamidino-2-phenylindole (DAPI) and annexin V/propidium iodide. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels were observed using fluorescence microscopy. Western blot analysis was used to quantify expression of proteins involved in apoptosis, cell cycle, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling. Pharmacokinetic and in vivo efficacy studies against Ehrlich ascites carcinoma (EAC) and Ehrlich solid tumor models were conducted using Swiss albino mice. RESULTS: IIIM-067 showed potent cytotoxicity and better selectivity than all other colchicine analogs screened in this study. The selective activity of IIIM-067 toward A549 cells was higher among other cancer cell lines, with a selectivity index (SI) value of 2.28. IIIM-067 demonstrated concentration- and time-dependent cytotoxicity against A549 cells with half-maximal inhibitory concentration values of 0.207, 0.150 and 0.106 µmol/L at 24, 48 and 72 h, respectively. It also had reduced toxicity to normal cells (SI > 1) than the parent compound colchicine (SI = 1). IIIM-067 reduced the clonogenic ability of A549 cells in a dose-dependent manner. IIIM-067 enhanced ROS production from 24.6% at 0.05 µmol/L to 82.1% at 0.4 µmol/L and substantially decreased the MMP (100% in control to 5.6% at 0.4 µmol/L). The annexin V-FITC assay demonstrated 78% apoptosis at 0.4 µmol/L. IIIM-067 significantly (P < 0.5) induced the expression of various intrinsic apoptotic pathway proteins, and it differentially regulated the PI3K/AKT/mTOR signaling pathway. Furthermore, IIIM-067 exhibited remarkable in vivo anticancer activity against the murine EAC model, with tumor growth inhibition (TGI) of 67.0% at a dose of 6 mg/kg (i.p.) and a reduced mortality compared to colchicine. IIIM-067 also effectively inhibited the tumor growth in the murine solid tumor model with TGI rates of 48.10%, 55.68% and 44.00% at doses of 5 mg/kg (i.p.), 6 mg/kg (i.p.) and 7 mg/kg (p.o.), respectively. CONCLUSION: IIIM-067 exhibited significant anticancer activity with reduced toxicity both in vitro and in vivo and is a promising anticancer candidate. However, further studies are required in clinical settings to fully understand its potential.

Antimicrobial and Cytotoxic Potential of Helminthosporin from Rumex abyssiniscus Jacq. Discovered as a Novel Source of Syringic Acid and Bis(2-ethyloctyl) Phthalate.

Rumex abyssinicus Jacq. is a perennial medicinal herb widely used in traditional medicine to treat many diseases. Phytochemicals of the plant were isolated using column chromatography and thin layer chromatography techniques. Extract, fractions and pure compounds were screened for antimicrobial activity against sensitive and multi-drug resistant microbes and their cytotoxicity was performed on different cancer cell lines. The mechanism of action of purified helminthosporin as well as the potent fraction containing a mixture of two compounds was assessed. Fraction R7C3 was the most potent antibacterial with the lowest MIC value of 0.12 µg/mL. Helminthosporin was the most potent compound with the lowest MIC value of 1.95 µg/mL. The compound was more potent than the antibiotic chloramphenicol against multi-drug resistant (MDR) bacteria with MIC equal to 16 µg/mL. The fraction and helminthosporin were shown to destroy the cell wall of the yeast and bacteria, and DNA fragmentation effect on the genome of Candida albicans and Bacillus cereus. Helminthosporin was the most cytotoxic compound with IC50 ˂ 10 µM. Fraction R7C3 showed the most potent cytotoxic effects on all cancer cell lines, with IC50 ranging from ˂1 to 4.35 ng/mL. Our study is the first report on the mechanism of action of helminthosporin, a potent candidate in the development of new drugs against multi-resistant bacteria and cancer cells. In addition, this study uncovered Rumex abyssinicus as a new source of syringic acid and bis(2-ethyloctyl) phthalate.

Plant growth promoting potential of butyl isobutyl phthalate and Streptomyces sp. from Rumex dentatus on rice.

Rice (Oryza sativa L.) is one of the most important staple foods consumed in many countries of the world. It is mostly consumed in developing countries where different chemical fertilizers are used to improve the productivity of the crop plant. In the present study, endophytic actinomycetes isolated from Rumex dentatus were identified morphologically and by scanning electron microscopy. Butyl isobutyl phthalate (BIBP) was isolated from the root endophyte Streptomyces sp. JR9 using column chromatography and HPLC methods. The compound was tested for its effect on rice seed germination. BIBP, extracts, and isolates were evaluated for their plant growth effect on rice in a growth chamber. Isolates were also screened in vitro for phosphate solubilization activity and enzyme production. Indole-3-acetic acid (IAA) and BIBP produced in extracts were quantified and detected using high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) methods, respectively. BIBP was found to increase the germination of rice seeds by 6 to 12% in treated samples and displayed potent effect at lowest concentration (0.437 µM). Both the compound and the extract depicted significant increases in almost all growth parameters at lowest concentration of 0.125 µg/mL and 62.5 µg/mL, respectively. BIBP also increased significantly shoot length, fresh root, fresh shoot, and dried shoot weight at high concentrations and was more potent than the standard phytohormone IAA. HPLC quantification showed 7.952 µg/mg and 0.371 µg/mg of IAA in extracts of Streptomyces sp. JR9 and the stem endophyte Streptomyces sp. KS3, respectively. IAA containing extract of JR9 increased significantly most growth parameters at lowest concentration (125 µg/mL). The extract of KS3 depicted significant increases in almost all growth parameters at high concentration (500 µg/mL). Our investigation showed that streptomycetes isolated from R. dentatus and BIBP are potent growth promoting agents and can be used in agriculture as bio-fertilizer to improve the growth and productivity of rice. KEY POINTS: • Butyl isobutyl phthalate (BIBP) isolated from endophytic Streptomyces sp. JR9 is a potent rice seed germination activator and promotes significantly the growth of rice • Isolated endophytes showed the ability to produce enzymes and phytohormone IAA • Isolates enhanced significantly the growth of rice.

Identification of plant-based multitargeted leads for Alzheimer's disease: In-vitro and in-vivo validation of Woodfordia fruticosa (L.) Kurz.

BACKGROUND: Alzheimer's disease (AD) is a complex neurodegenerative disease with no availability of disease-modifying therapeutics. The complex etiology and recent failures in clinical trials indicate the need for multitargeted agents. PURPOSE: The present study aims to discover new plant-based multitargeted anti-AD leads. METHODS: A library of plant extracts was screened for inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1). The secondary metabolites of active extracts were also tested, followed by enzyme-kinetics and molecular modeling to understand the mechanism of inhibition. The most active extract was investigated for in-vivo anti-dementia activity in behavioral mice models. RESULTS: Among the library of 105 extracts, Woodfordia fruticosa (SBE-80) and Bergenia ciliata (SBE-65) extracts displayed significant inhibition of all three enzymes. Gallic acid, one of the constituents of both plants, shows moderate inhibition of AChE and BACE-1. Catechin-3-O-gallate (CG), another constituent of SBE-65, inhibits EeAChE, rHuAChE, and eqBChE with IC50's of 29.9, 1.77, and 8.4 µM, respectively; along with a mild-inhibition of BACE-1. Ellagic acid, the constituent of SBE-80, inhibits BACE-1 with an IC50 value of 16 µM. The W. fruticosa extract SBE-80 at the dose of 25 mg/kg QD × 9 (PO) displayed memory-enhancing activity in Morris Water Maze and Passive Avoidance Test in Swiss albino mice. Treatment with SBE-80 also inhibits AChE in-vivo; whereas, a non-significant decrease in the serum TBARS was observed. CONCLUSION: W. fruticosa is identified for the first time as an anti-AD lead candidate. The in-vitro and in-vivo data presented herein and the documented safety profile of W. fruticosa indicate its strong potential for preclinical development as a botanical drug for dementia/AD.

Natural Products in Mitigation of SARS CoV Infections.

Severe acute respiratory syndrome (SARS) is a critical respiratory disease caused by coronaviruses (CoV). The available antiviral agents or host-specific antiinflammatory therapies are the principal treatment modalities, with drug-repurposing as the most viable approach to timely tackle the CoV pandemic. Though these approaches are successful to some extent in reducing the mortality rate, however, it is too far to see a complete escape from the current SARS CoV-2 pandemic. Plants are the primary source of diet, dietary supplements, botanical drugs, and natural products (NPs). It has been well accepted and proved via several scientific studies that plant-based therapies play a vital role in managing such infections. The faulty immune system (compromised innate immunity or aberrant immune activation) determines the severity of the respiratory distress in CoV-2 infected patients. Natural products intervene at various stages of the virus replication cycle, including inhibition of virus entry into the host cells, inhibition of serine/ cysteine proteases, RNA-dependent RNA polymerase (RdRp) or helicase. Besides, several natural products or plant-based dietary supplements have a unique ability to strengthen the immune system or alleviate the hyper-inflammatory condition. Many plant-based formulations, dietary supplements, and NPs are being investigated in clinical trials in CoV-2 infected patients, and few have already shown positive results. The review has unearthed several NP leads for medicinal chemistry programs as well as some having direct opportunity of repurposing in SARS CoV infections.

Evaluation of rohitukine-enriched fraction of Dysoxylum binectariferum Hook.f. (leaves) as anti-arthritic phytopharmaceutical candidate: Chemical standardization, in-vivo validation, formulation development and oral pharmacokinetics.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis is a chronic inflammatory disease of joints. Dysoxylum binectariferum Hook.f (Family: Meliaceae) is a Indian medicinal plant which is traditionally being used to heal inflammation of joints. AIM OF THE STUDY: This work was aimed to carry out chemical standardization, in-vitro/in-vivo validation, oral pharmacokinetics and formulation development of anti-arthritic botanical lead, the rohitukine-enriched fraction of D. binectariferum. MATERIALS AND METHODS: The rohitukine-enriched fraction of D. binectariferum was standardized using four chemical markers and was checked for microbial load, heavy metal content, aflatoxins and pesticides. Its in-vitro inhibitory effect on the lipopolysaccharide (LPS) induced production of pro-inflammatory cytokines TNF-α and IL-6 was studied in THP-1 cells. The in-vivo anti-arthritic activity was investigated in collagen-induced arthritis model in DBA/1J mice. The sustained release capsule formulation was developed and characterized for physicochemical and pharmacokinetic properties. RESULTS: Rohitukine and schumaniofioside A were found to be major chemical constituents of the botanical lead. The rohitukine-enriched fraction of D. binectariferum significantly reduced the production of both pro-inflammatory cytokines TNF-α and IL-6 (>50% inhibition at 3.12 µg/mL) in THP-1 cells. In LPS-treated wild-type mice model, the rohitukine-enriched fraction at 200 mg/kg (PO, QD) completely reduced serum TNF-α levels. In transgenic mice model (collagen-induced arthritis in DBA/1J mice), rohitukine-enriched fraction at 100 mg/kg (PO, QD) dose has resulted in >75% reduction of TNF-α/IL-6 serum levels, 68% reduction in anti-mouse type II collagen IgG1 antibody levels, decreased joint proteoglycan loss and reduced paw edema in DBA/1J mice. The sustained release capsule formulation of rohitukine-enriched fraction showed sustained-release of rohitukine over the period of 24 h, and resulted in an improved plasma-exposure of rohitukine in SD rats. CONCLUSIONS: The data presented herein demonstrated anti-arthritic potential of rohitukine-enriched fraction of D. binectariferum and this study will serve as the benchmark for further research on this botanical lead and developed sustained release capsule formulation.

Discovery of benzo[cd]indol-2-one and benzylidene-thiazolidine-2,4-dione as new classes of NLRP3 inflammasome inhibitors via ER-ß structure based virtual screening.

The structure-guided virtual screening (VS) has proved to be successful strategy in identification of new scaffolds for biological targets. The overactivity of NLRP3 inflammasome has been implicated in variety of inflammatory diseases including Alzheimer's disease. The up-regulation of estrogen-receptor ß (ER-ß) activity has been directly linked with inhibition of NLRP3 inflammasome activity. In the present study, we report discovery of new NLRP3 inflammasome inhibitors via ER-ß crystal structure (PDB: 5TOA) guided virtual screening of 20,000 compound library. For experimental validation, top 10 ligands were selected based on structure novelty, docking score, prime MMGB/SA binding affinity and interaction pattern analysis. Amongst the tested compounds, three thiazolidin-4-ones IIIM-1268, IIIM-1269 and IIIM-1270 and benzo[cd]indol-2-one IIIM-1266 have shown 73, 69, 75 and 77% suppression of IL-1ß release in mouse macrophages (J774A.1 cells) at 10 µM. Benzylidene-thiazolidine-2,4-diones IIIM-1268 and IIIM-1270 inhibited IL-1ß release with IC50 of 2.3 and 3.5 µM and also significantly decreased the protein expression level of mature form of IL-1ß in western-blot analysis. IIIM-1266 and IIIM-1270 displayed bidentate H-bonding with Arg 346 and Glu 305 residues in the active site of ER-ß; and they also strongly occupied the ADP-binding site of NLRP3 protein. The results presented herein, indicate that ER-ß guided VS can be successfully used to identify new NLRP3 inflammasome inhibitors, which may have potential in the development of novel anti-Alzheimer agents.

Binary and ternary solid dispersions of an anticancer preclinical lead, IIIM-290: In vitro and in vivo studies.

The objective of this study was to formulate an anticancer preclinical lead, IIIM-290, loaded in solid dispersions to enhance its solubility, dissolution, and oral pharmacokinetics. IIIM-290 is an in-house preclinical anticancer lead prepared by semisynthetic modification of the natural product rohitukine. It is an orally bioavailable Cdk inhibitor showing efficacy in xenograft models of pancreatic, colon and leukemia cancer. It demonstrated in vivo efficacy at a relatively higher dose owing to its poor aqueous solubility (~8.6 µg/mL). Binary and ternary solid dispersions containing PVP K-30, xanthan gum, and PEG-PPG-PEG were selected after solubility screening of various hydrophilic polymers. Several formulations with varying ratios of polymers, alone and in combination, were prepared and investigated for their effects on the solubility enhancement of IIIM-290. The binary solid dispersion VKB-SD75, prepared with PVP K-30 at the ratio of 1:4 w/w, was identified as the optimized composition that displayed 17-fold improvement in the aqueous solubility of IIIM-290. VKB-SD75 was scaled up to a 100-g scale. IIIM-290 and VKB-SD75 were evaluated for DSC, p-XRD, FTIR, 1H NMR, SEM, in vitro dissolution, and oral pharmacokinetics, as well as for in vivo anticancer activity in the Ehrlich solid tumor model. The oral administration of VKB-SD75 in BALB/c mice resulted in a 1.9-fold improvement in plasma exposure. These findings also correlated well when the formulation was administered to mice in the Ehrlich solid tumor model. The newly developed solid dispersion is expected to reduce the dose of IIIM-290 by ~40-50% in preclinical and clinical studies.

Identification of embelin, a 3-undecyl-1,4-benzoquinone from Embelia ribes as a multitargeted anti-Alzheimer agent.

Beta-secreatse (BACE-1) and cholinesterases are clinically validated targets of Alzheimer's disease (AD), for which natural products have provided immense contribution. The multifaceted nature of AD signifies the need of multitargeted agents to tackle this disease. In the search of new natural products as dual BACE-1/cholinesterase inhibitors, a library of pure natural products was screened for inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE-1. The screening efforts have identified 1,4-benzoquinone "embelin," a natural product derived from Embelia ribes displaying inhibition of all three enzymes, with IC50 values of 2.5, 5.4, and 2.1 µM, respectively. This screen has also identified isoquinoline alkaloids papaverine and L-tetrahydropalmatine as AChE inhibitors. Kinetic study has shown that embelin inhibits EeAChE and EqBChE with ki values of 4.59 and 0.57 µM, in an uncompetitive and noncompetitive manner, respectively. The interactions of embelin with allosteric peripheral anionic site of cholinesterases, has further supported the results of kinetic study. Embelin has also enhanced the activity of P-gp in LS-180 cells, the efflux pump which is involved in the clearance of amyloid-ß from AD brain. Further, the cell viability study in neuronal cell line has indicated the excellent therapeutic window of embelin. These results are indicative of the fact that embelin is a multitargeted agent playing role in stopping the formation of amyloid-ß oligomers (via inhibition of BACE-1), improves cholinergic-transmission (via inhibition of AChE/BChE) and increases amyloid-ß clearance (via P-gp induction).

Discovery and preclinical development of IIIM-160, a Bergenia ciliata-based anti-inflammatory and anti-arthritic botanical drug candidate.

OBJECTIVE: Bergenia ciliata (Haw.) Sternb. is used in the Indian traditional system of medicine to treat various ailments including rheumatism and to heal wounds. The objective of the present study was to perform a preclinical characterization of the B. ciliata-based botanical extract IIIM-160. METHODS: IIIM-160 was chemically standardized and analyzed for heavy metal content, aflatoxins, pesticides and microbial load. The in vitro and in vivo efficacies were determined in suitable models of inflammation, arthritis and nociception. An acute oral toxicity study was performed in Swiss albino mice. A suitable oral formulation was developed and characterized. RESULTS: Bergenin was found to be the major component (9.1% w/w) of IIIM-160. The botanical lead displayed inhibition of lipopolysaccharide-induced production of proinflammatory cytokines in THP-1 cells, with selectivity toward interleukin-6 (IL-6) and had an excellent safety-window. It showed anti-inflammatory, anti-arthritic and antinociceptive activity in animal models and was not toxic at oral doses up to 2 g/kg in Swiss-albino mice. The gastroretentive, sustained-release capsule formulation showed sustained-release of the bergenin over the period of 24 h, resulting in improved plasma-exposure of bergenin in Sprague-Dawley rats. CONCLUSION: The dual-activity of IL-6 inhibition and antinociception marks the suitability of IIIM-160 for treating rheumatoid arthritis. This study will serve as the benchmark for further research on this botanical formulation.

Furanoflavones pongapin and lanceolatin B blocks the cell cycle and induce senescence in CYP1A1-overexpressing breast cancer cells.

Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is induced (a) by polycyclic aromatic hydrocarbons (PAHs) which can be metabolized by CYP1A1 to carcinogens, and (b) in majority of breast cancers. Hence, phytochemicals or dietary flavonoids, if identified as CYP1A1 inhibitors, may help in preventing PAH-mediated carcinogenesis and breast cancer. Herein, we have investigated the cancer chemopreventive potential of a flavonoid-rich Indian medicinal plant, Pongamia pinnata (L.) Pierre. Methanolic extract of its seeds inhibits CYP1A1 in CYP1A1-overexpressing normal human HEK293 cells, with IC50 of 0.6 µg/mL. Its secondary metabolites, the furanoflavonoids pongapin/lanceolatin B, inhibit CYP1A1 with IC50 of 20 nM. Although the furanochalcone pongamol inhibits CYP1A1 with IC50 of only 4.4 µM, a semisynthetic pyrazole-derivative P5b, has ∼10-fold improved potency (IC50, 0.49 µM). Pongapin/lanceolatin B and the methanolic extract of P. pinnata seeds protect CYP1A1-overexpressing HEK293 cells from B[a]P-mediated toxicity. Remarkably, they also block the cell cycle of CYP1A1-overexpressing MCF-7 breast cancer cells, at the G0-G1 phase, repress cyclin D1 levels and induce cellular-senescence. Molecular modeling studies demonstrate the interaction pattern of pongapin/lanceolatin B with CYP1A1. The results strongly indicate the potential of methanolic seed-extract and pongapin/lanceolatin B for further development as cancer chemopreventive agents.

Identification of karanjin isolated from the Indian beech tree as a potent CYP1 enzyme inhibitor with cellular efficacy via screening of a natural product repository.

CYP1A1 is thought to mediate carcinogenesis in oral, lung and epithelial cancers. In order to identify a CYP1A1 inhibitor from an edible plant, 394 natural products in the IIIM's natural product repository were screened, at 10 µM concentration, using CYP1A1-Sacchrosomes™ (i.e. microsomal enzyme isolated from recombinant baker's yeast). Twenty-seven natural products were identified that inhibited 40-97% of CYP1A1's 7-ethoxyresorufin-O-deethylase activity. The IC50 values of the 'hits', belonging to different chemical scaffolds, were determined. Their selectivity was studied against a panel of 8 CYP-Sacchrosomes™. In order to assess cellular efficacy, the 'hits' were screened for their capability to inhibit CYP enzymes expressed within live recombinant human embryonic kidney (HEK293) cells from plasmids encoding specific CYP genes (1A2, 1B1, 2C9, 2C19, 2D6, 3A4). Isopimpinellin (IN-475; IC50, 20 nM) and karanjin (IN-195; IC50, 30 nM) showed the most potent inhibition of CYP1A1 in human cells. Isopimpinellin is found in celery, parsnip, fruits and in the rind and pulp of limes whereas different parts of the Indian beech tree, which contain karanjin, have been used in traditional medicine. Both isopimpinellin and karanjin negate the cellular toxicity of CYP1A1-mediated benzo[a]pyrene. Molecular docking and molecular dynamic simulations with CYP isoforms rationalize the observed trends in the potency and selectivity of isopimpinellin and karanjin.

Discovery and Preclinical Development of IIIM-290, an Orally Active Potent Cyclin-Dependent Kinase Inhibitor.

Rohitukine (1), a chromone alkaloid isolated from Indian medicinal plant Dysoxylum binectariferum, has inspired the discovery of flavopiridol and riviciclib, both of which are bioavailable only via intravenous route. With the objective to address the oral bioavailability issue of this scaffold, four series of rohitukine derivatives were prepared and screened for Cdk inhibition and cellular antiproliferative activity. The 2,6-dichloro-styryl derivative IIIM-290 (11d) showed strong inhibition of Cdk-9/T1 (IC50 1.9 nM) kinase and Molt-4/MIAPaCa-2 cell growth (GI50 < 1.0 µM) and was found to be highly selective for cancer cells over normal fibroblast cells. It inhibited the cell growth of MIAPaCa-2 cells via caspase-dependent apoptosis. It achieved 71% oral bioavailability with in vivo efficacy in pancreatic, colon, and leukemia xenografts at 50 mg/kg, po. It did not have CYP/efflux-pump liability, was not mutagenic/genotoxic or cardiotoxic, and was metabolically stable. The preclinical data presented herein indicates the potential of 11d for advancement in clinical studies.

Preclinical Development of Crocus sativus-Based Botanical Lead IIIM-141 for Alzheimer's Disease: Chemical Standardization, Efficacy, Formulation Development, Pharmacokinetics, and Safety Pharmacology.

Crocus sativus L. (family: Iridaceae) has been documented in traditional medicine with numerous medicinal properties. Recently, we have shown that C. sativus extract (IIIM-141) displays promising efficacy in a genetic mice (5XFAD) model of Alzheimer's disease (AD) (ACS Chem. Neurosci. 2017, 16, 1756). To translate the available traditional knowledge and the scientifically validated results into modern medicine, herein we aimed to carry out its preclinical development. IIIM-141 is primarily a mixture of crocins containing trans-4-GG-crocin (36 % w/w) as the principal component. The in vitro studies show that IIIM-141 has protective as well as therapeutic properties in assays related to AD. It induces the expression of P-gp, thereby enhancing the amyloid-ß clearance from an AD brain. It also inhibits NLRP3 inflammasome and protects SH-SY5Y cells against amyloid-ß- and glutamate-induced neurotoxicities. In behavioral models, it decreased the streptozotocin-induced memory impairment in rats and recovered the scopolamine-induced memory deficit in Swiss albino mice at 100 mg/kg dose. The acute oral toxicity study shows that IIIM-141 is safe up to the dose of 2000 mg/kg, with no effect on the body weight and on the biochemical/hematological parameters of the rats. The repeated oral administration of IIIM-141 for 28 days at 100 mg/kg dose did not cause any preterminal deaths and abnormalities in Wistar rats. The pharmacokinetic analysis indicated that after oral administration of IIIM-141, the majority of crocin gets hydrolyzed to its aglycone crocetin. The sustained release (SR) capsule formulation was developed, which showed an improved in vitro dissolution profile and a significantly enhanced plasma exposure in the pharmacokinetic study. The SR formulation resulted in 3.3-fold enhancement in the area under the curve of crocetin and doubling of the crocetin/crocin ratio in plasma compared with the extract. The data presented herein will serve as the benchmark for further research on this botanical candidate.

Glycyrrhiza glabra extract and quercetin reverses cisplatin resistance in triple-negative MDA-MB-468 breast cancer cells via inhibition of cytochrome P450 1B1 enzyme.

The development of multi-drug resistance to existing anticancer drugs is one of the major challenges in cancer treatment. The over-expression of cytochrome P450 1B1 enzyme has been reported to cause resistance to cisplatin. With an objective to discover cisplatin-resistance reversal agents, herein, we report the evaluation of Glycyrrhiza glabra (licorice) extracts and its twelve chemical constituents for inhibition of CYP1B1 (and CYP1A1) enzyme in Sacchrosomes and live human cells. The hydroalcoholic extract showed potent inhibition of CYP1B1 in both Sacchrosomes as well as in live cells with IC50 values of 21 and 16 µg/mL, respectively. Amongst the total of 12 constituents tested, quercetin and glabrol showed inhibition of CYP1B1 in live cell assay with IC50 values of 2.2 and 15 µM, respectively. Both these natural products were found to be selective inhibitors of CYP1B1, and does not inhibit CYP2 and CYP3 family of enzymes (IC50 > 20 µM). The hydroalcoholic extract of G. glabra and quercetin (4) showed complete reversal of cisplatin resistance in CYP1B1 overexpressing triple negative MDA-MB-468 breast cancer cells. The selective inhibition of CYP1B1 by quercetin and glabrol over CYP2 and CYP3 family of enzymes was studied by molecular modeling studies.

Crocus sativus Extract Tightens the Blood-Brain Barrier, Reduces Amyloid ß Load and Related Toxicity in 5XFAD Mice.

Crocus sativus, commonly known as saffron or Kesar, is used in Ayurveda and other folk medicines for various purposes as an aphrodisiac, antispasmodic, and expectorant. Previous evidence suggested that Crocus sativus is linked to improving cognitive function in Alzheimer's disease (AD) patients. The aim of this study was to in vitro and in vivo investigate the mechanism(s) by which Crocus sativus exerts its positive effect against AD. The effect of Crocus sativus extract on Aß load and related toxicity was evaluated. In vitro results showed that Crocus sativus extract increases the tightness of a cell-based blood-brain barrier (BBB) model and enhances transport of Aß. Further in vivo studies confirmed the effect of Crocus sativus extract (50 mg/kg/day, added to mice diet) on the BBB tightness and function that was associated with reduced Aß load and related pathological changes in 5XFAD mice used as an AD model. Reduced Aß load could be explained, at least in part, by Crocus sativus extract effect to enhance Aß clearance pathways including BBB clearance, enzymatic degradation and ApoE clearance pathway. Furthermore, Crocus sativus extract upregulated synaptic proteins and reduced neuroinflammation associated with Aß pathology in the brains of 5XFAD mice. Crocin, a major active constituent of Crocus sativus and known for its antioxidant and anti-inflammatory effect, was also tested separately in vivo in 5XFAD mice. Crocin (10 mg/kg/day) was able to reduce Aß load but to a lesser extent when compared to Crocus sativus extract. Collectively, findings from this study support the positive effect of Crocus sativus against AD by reducing Aß pathological manifestations.

Identification of Potent and Selective CYP1A1 Inhibitors via Combined Ligand and Structure-Based Virtual Screening and Their in Vitro Validation in Sacchrosomes and Live Human Cells.

Target structure-guided virtual screening (VS) is a versatile, powerful, and inexpensive alternative to experimental high-throughput screening (HTS). To discover potent CYP1A1 enzyme inhibitors for cancer chemoprevention, a commercial library of 50 000 small molecules was utilized for VS guided by both ligand and structure-based strategies. For experimental validation, 300 ligands were proposed based on combined analysis of fitness scores from ligand based e-pharmacophore screening and docking score, prime MMGB/SA binding affinity and interaction pattern analysis from structure-based VS. These 300 compounds were screened, at 10 µM concentration, for in vitro inhibition of CYP1A1-Sacchrosomes (yeast-derived microsomal enzyme) in the ethoxyresorufin-O-de-ethylase assay. Thirty-two compounds displayed >50% inhibition of CYP1A1 enzyme activity at 10 µM. 2-Phenylimidazo-[1,2-a]quinoline (5121780, 119) was found to be the most potent with 97% inhibition. It also inhibited ∼95% activity of CYP1B1 and CYP1A2, the other two CYP1 enzymes. The compound 5121780 (119) showed high selectivity toward inhibition of CYP1 enzymes with respect to CYP2 and CYP3 enzymes (i.e., there was no detectable inhibition of CYP2D6/CYP2C9/CYP2C19 and CYP3A4 at 10 µM). It was further investigated in live CYP-expressing human cell system, which confirmed that compound 5121780 (119) potently inhibited CYP1A1, CYP1A2, CYP1B1 enzymes with IC50 values of 269, 30, and 56 nM, respectively. Like in Sacchrosomes, inhibition of CYP2D6/CYP2C9/CYP2C19 and CYP3A4 enzymes, expressed within live human cells, could hardly be detected at 10 µM. The compound 119 rescued CYP1A1 overexpressing HEK293 cells from CYP1A1 mediated benzo[a]pyrene (B[a]P) toxicity and also overcame cisplatin resistance in CYP1B1 overexpressing HEK293 cells. Molecular dynamics simulations of 5121780 (119) with CYP1 enzymes was performed to understand the interaction pattern to CYP isoforms. Results indicate that VS can successfully be used to identify promising CYP1A1 inhibitors, which may have potential in the development of novel cancer chemo-preventive agents.

Antioxidant and antiproliferative activity of indigocarpan, a pterocarpan from Indigofera aspalathoides.

OBJECTIVES: This study aimed to investigate the anticancer potential of indigocarpan (1), a pterocarpan isolated from Indigofera aspalathoides, a plant found in India which has been used in Ayurveda for centuries for the treatment of oedematous tumours. METHODS: The antiproliferative activity in a panel of four human cancer cell lines was studied. The mechanism of its antiproliferative activity in human colorectal adenocarcinoma LS174T cells was investigated in detail. KEY FINDINGS: Indigocarpan (1) showed antiproliferative activity in a panel of four human cancer cell lines with IC50 s ranging from 180 to 250 µm. Indigocarpan induces p53-dependent p21 upregulation and apoptosis in LS174T cells, upregulates p53 and p21(WAF1) protein levels, enhances cleavage of caspase-3 and downregulates cyclin D1, cyclin B1 and PCNA protein levels, indicating its role in modulating cell cycle progression. Indigocarpan also exhibited a strong antioxidative effect in LS174T cells. CONCLUSIONS: Along with the antiproliferative capacity, the strong antioxidative property of the compound makes it a promising candidate for further development as an anticancer and chemopreventive compound.

A chromatography-free isolation of rohitukine from leaves of Dysoxylum binectariferum: Evaluation for in vitro cytotoxicity, Cdk inhibition and physicochemical properties.

Rohitukine is a chromone alkaloid isolated from an Indian medicinal plant Dysoxylum binectariferum. This natural product has led to the discovery of two clinical candidates (flavopiridol and P276-00) for the treatment of cancer. Herein, for the first time we report an efficient protocol for isolation and purification of this precious natural product in a bulk-quantity from leaves (a renewable source) of D. binectariferum (>98% purity) without use of chromatography or any acid-base treatment. Despite of the fact that this scaffold has reached up to clinical stage, particularly for leukemia; however the antileukemic activity of a parent natural product has never been investigated. Furthermore, rohitukine has never been studied for cyclin-dependent kinase (Cdk) inhibition, kinase profiling and for its experimental physicochemical properties. Thus, herein, we report in vitro cytotoxicity of rohitukine in a panel of 20 cancer cell lines (including leukemia, pancreatic, prostate, breast and CNS) and 2 normal cell lines; kinase profiling, Cdk2/9 inhibition, and physicochemical properties (solubility and stability in biological medias, pKa, LogP, LogD). In cytotoxicity screening, rohitukine displayed promising activity in HL-60 and Molt-4 (leukemia) cell lines with GI50 of 10 and 12µM, respectively. It showed inhibition of Cdk2/A and Cdk9/T1 with IC50 values of 7.3 and 0.3µM, respectively. The key interactions of rohitukine with Cdk9 was also studied by molecular modeling. Rohitukine was found to be highly water soluble (Swater=10.3mg/mL) and its LogP value was -0.55. The ionization constant of rohitukine was found to be 5.83. Rohitukine was stable in various biological media's including rat plasma. The data presented herein will help in designing better anticancer agents in future.