Experimental and clinical data collected over the last 25 years indicate that the anti-inflammatory, antipyretic, and analgesic properties of aspirin, ibuprofen, and other nonsteroidal anti-inflammatory drugs result from their ability to block prostaglandin (PG) synthesis. Prostaglandin endoperoxide H synthase (PGHSs) catalyze the conversion of arachidonate to PGH2. These enzymes have two activities: a cyclooxygenase activity that incorporates two molecules of oxygen into arachidonate to form PGG2 and a peroxidase activity that reduces the 15-hydroperoxide group of PGG2 to form PGH2. PGHSs are membrane proteins, and therefore preparation of microsomal membranes is a simple way to purify the enzymes partially before use in inhibitor assays. While not absolutely essential, isolation of membranes results in about a tenfold purification of PGHSs, and removes many of the soluble and nuclear proteins that may interfere with measurements of cyclooxygenase activity and determinations of inhibition constants.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor found in fluids lining mucosal surfaces. In addition to its primary function as an antiprotease, SLPI may also influence cellular functions associated with en- zyme synthesis and retroviral infection. In this study, SLPI was examined for its effect on signaling events involved in the production of matrix metalloproteinases (MMPs) by monocytes. Addition of SLPI before stimulation with con- canavalin A or LPS resulted in a significant inhibition of monocyte prostaglandin H synthase-2 (PGHS-2), a pivotal enzyme in the PGE 2 -cAMP dependent pathway of mono- cyte MMP synthesis. Suppression of PGHS-2 was detected with 0.1 m g/ml of SLPI with a substantial inhibition at 1 and 10 m g/ml. Attenuation of PGHS-2 by SLPI was accompa- nied by decreased production of PGE 2 resulting in the sup- pression of interstitial collagenase (MMP-1) and gelatinase B (MMP-9) that was reversed by PGE 2 or Bt 2 cAMP. The in- hibitory effect of SLPI was largely independent of its anti- protease activity because SLPI muteins, with significantly lower antiprotease activity, also suppressed the induction of PGHS-2 and MMPs. The inhibitory effects of SLPI did not involve the modulation of monokine production since TNF- a and IL-10 were unaffected. These findings demonstrate that SLPI also functions as a potent antiinflammatory agent by interfering with the signal transduction pathway leading to monocyte MMP production. ( J. Clin. Invest. 1997. 99:894-

Efforts to apply nanotechnology in cancer have focused almost exclusively on the delivery of cytotoxic drugs to improve therapeutic index. There has been little consideration of molecularly targeted agents, in particular kinase inhibitors, which can also present considerable therapeutic index limitations. We describe the development of Accurin polymeric nanoparticles that encapsulate the clinical candidate AZD2811, an Aurora B kinase inhibitor, using an ion pairing approach. Accurins increase biodistribution to tumor sites and provide extended release of encapsulated drug payloads. AZD2811 nanoparticles containing pharmaceutically acceptable organic acids as ion pairing agents displayed continuous drug release for more than 1 week in vitro and a corresponding extended pharmacodynamic reduction of tumor phosphorylated histone H3 levels in vivo for up to 96 hours after a single administration. A specific AZD2811 nanoparticle formulation profile showed accumulation and retention in tumors with minimal impact on bone marrow pathology, and resulted in lower toxicity and increased efficacy in multiple tumor models at half the dose intensity of AZD1152, a water-soluble prodrug of AZD2811. These studies demonstrate that AZD2811 can be formulated in nanoparticles using ion pairing agents to give improved efficacy and tolerability in preclinical models with less frequent dosing. Accurins specifically, and nanotechnology in general, can increase the therapeutic index of molecularly targeted agents, including kinase inhibitors targeting cell cycle and oncogenic signal transduction pathways, which have to date proved toxic in humans.

Background: BIND-2206 Accurins are novel polymeric nanoparticles encapsulating MK-2206, a Merck AKT inhibitor in phase 2 trials. MK-2206 targets the phosphatidylinositol 3-kinase (PI3K) pathway via AKT inhibition and has demonstrated therapeutic efficacy in the treatment of cancer albeit with dose limiting toxicities. Accurins have shown promise for targeting oncology agents preferentially to tumors, while limiting systemic exposure. To determine if encapsulation of MK-2206 improves therapeutic index, studies were performed to evaluate pharmacokinetics (PK), pharmacodynamics (PD), tolerability and anti-tumor activity compared to parent MK-2206. Materials and Methods: PK of four Accurin formulations with varying in vitro drug release rates was evaluated. Based on these data, two lead Accurins (BIND-2206C and BIND-2206D) were selected for further characterization. Since AKT inhibitors are documented to induce hyperglycemia in mice, blood glucose levels were also evaluated after acute administration of BIND-2206 Accurins or parent MK-2206 as a measure of tolerability. Using a panel of human tumor xenografts (VCaP, SK-OV-3 and BT-474) anti-tumor activity of the Accurins was assessed in mice. When tumors were established mice were dosed orally with parent MK-2206 or intravenously with BIND-2206 Accurins two or three times per week on a three week cycle. Tumor volume was measured post treatment to determine anti-tumor activity. Following final drug administration, tumors were collected for PK and PD evaluation. Extent and duration of tumor target inhibition was evaluated by measuring pAKT and total AKT using a mesoscale discovery method and was correlated to tumor MK-2206 levels. Results: Accurins BIND-2206C and BIND-2206D significantly enhanced the PK profile of parent MK-2206 by increasing Cmax (33 fold) and AUC (222 fold) and decreasing clearance (38 fold) in mice. In addition, both BIND-2206 Accurins improved tolerability as demonstrated by no hyperglycemic response compared to a 300% increase in blood glucose in nude mice treated with parent MK-2206. In the VCaP human prostate cancer tumor model, BIND-2206C inhibited tumor growth by 88% and BIND-2206D induced 16% tumor regression showing significant enhancement of anti-tumor efficacy compared to 55% tumor growth inhibition achieved by parent MK-2206. This correlated with high tumor exposure at 72 hours and prolonged tumor PD at 96 hours post dose for both Accurins compared to parent MK-2206. A similar increase in tumor exposure and prolonged PD response resulted in enhanced anti-tumor efficacy for BIND-2206 Accurins in the SK-OV-3 human ovarian cancer tumor model. In the BT-474 HER2 over-expressing human breast cancer tumor model, BIND-2206 Accurins did not improve efficacy compared to parent drug, although there was significant and prolonged tumor PD correlating with enhanced tumor PK. This suggests that anti-tumor efficacy is model specific and a combination strategy in the BT-474 model may be advantageous. Conclusions: BIND-2206 Accurins showed differentiated PK, increased tumor exposure, prolonged duration of target inhibition, superior efficacy and enhanced tolerability compared to parent MK-2206. These data suggest that nanoparticle formulations of the AKT inhibitor, MK-2206, may provide improved tolerability and therapeutic efficacy in a clinical setting. Citation Format: Louise Cadzow, Michael H. Lam, Young Ho Song, Maria Figueiredo, Hong Wang, David De Witt, Vincenzo Pucci, Jan-Rung Mo, Eric Lewis-Clark, Heidi Ferguson, Marian Gindy, Susan Low, Steve Zale, Jeff Hrkach, Caroline McGregor, Brian J. Long. Accurins improve the pharmacokinetics, pharmacodynamics, tolerability and anti-tumor activity of the AKT inhibitor MK-2206. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C197.

Abstract The prodrug, Barasertib (AZD1152) is a potent selective inhibitor of the Aurora B kinase which plays a pivotal role in chromosomal segregation during the cell cycle. In a Phase 2 randomised study in elderly AML patients unfit for intensive therapy, Barasertib showed a statistically significant improvement in Overall Complete Response Rate and a trend for improved Overall Survival as a continuous infusion over 7 days (Kantarjian et al, Cancer 2013;119:2611-2619) . The Dose Limiting Toxicities were neutropenia and stomatitis. AccurinTM nanoparticle formulations of the active metabolite, AZD1152-hQPA, have been developed utilizing medicinal nanoengineering to optimize critical nanoparticle properties such that a short infusion has the potential for extended target cover and an improved therapeutic index from increased biodistribution to the tumour site. In nude rats bearing SW620 tumours, AZD1152-hQPA Accurins gave extended target cover leading to sustained pharmacodynamic (PD) inhibition of phospho-histone H3 in the tumour relative to Barasertib at the same dose. Interestingly the Accurins gave a differentiated PD profile with maximal PD seen at later time points. This profile led to improved anti-tumour effects when AZD1152-hQPA Accurins were dosed intravenously at 25mg/kg on day 1 and 3, compared to Barasertib dosed at 25mg/kg on days 1, 2, 3, 4. Significantly, one of the AZD1152-hQPA Accurins had minimal impact on bone marrow pathology, demonstrating the potential for increased efficacy and lower toxicity at half the dose intensity of Barasertib in this preclinical model. The impact on efficacy of different dose intensities and schedules with this AZD1152-hQPA Accurin has also been explored in the equivalent nude mouse SW620 model. Together these studies demonstrate that by careful selection of formulation parameters, AZD1152-hQPA can be formulated with an extended release profile able to deliver improved efficacy and tolerability in pre-clinical models. AZD1152-hQPA Accurins offer the potential to overcome the therapeutic index challenges which previously limited the clinical development of agents in the class. Citation Format: Simon T. Barry, Elaine Cadogan, Rajesh Odedra, Susan Ashton, John Foster, Young Ho Song, Susan Low, Paula Taylor, Rebecca Ellston, Urszula Polanska, Joanne Wilson, Colin Howes, Dawn Trueman, Mike Walker, David De Witt, Marianne Ashford, Jeff Hrkach, Phil Jewsbury. AZD1152-hQPA Accurins: Nanoparticle formulations showing extended release and the potential for improved therapeutic index. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5409. doi:10.1158/1538-7445.AM2014-5409

We describe the development and clinical translation of a targeted polymeric nanoparticle (TNP) containing the chemotherapeutic docetaxel (DTXL) for the treatment of patients with solid tumors. DTXL-TNP is targeted to prostate-specific membrane antigen, a clinically validated tumor antigen expressed on prostate cancer cells and on the neovasculature of most nonprostate solid tumors. DTXL-TNP was developed from a combinatorial library of more than 100 TNP formulations varying with respect to particle size, targeting ligand density, surface hydrophilicity, drug loading, and drug release properties. Pharmacokinetic and tissue distribution studies in rats showed that the NPs had a blood circulation half-life of about 20 hours and minimal liver accumulation. In tumor-bearing mice, DTXL-TNP exhibited markedly enhanced tumor accumulation at 12 hours and prolonged tumor growth suppression compared to a solvent-based DTXL formulation (sb-DTXL). In tumor-bearing mice, rats, and nonhuman primates, DTXL-TNP displayed pharmacokinetic characteristics consistent with prolonged circulation of NPs in the vascular compartment and controlled release of DTXL, with total DTXL plasma concentrations remaining at least 100-fold higher than sb-DTXL for more than 24 hours. Finally, initial clinical data in patients with advanced solid tumors indicated that DTXL-TNP displays a pharmacological profile differentiated from sb-DTXL, including pharmacokinetics characteristics consistent with preclinical data and cases of tumor shrinkage at doses below the sb-DTXL dose typically used in the clinic.

Tea prepared from the aerial parts of Antigonon leptopus is used as a remedy for cold and pain relief in many countries. In this study, A. leptopus tea, prepared from the dried aerial parts, was evaluated for lipid peroxidation (LPO) and cyclooxygenase (COX-1 and COX-2) enzyme inhibitory activities. The tea as a dried extract inhibited LPO, COX-1 and COX-2 enzymes by 78%, 38% and 89%, respectively, at 100 μg/mL. Bioassay-guided fractionation of the extract yielded a selective COX-2 enzyme inhibitory phenolic aldehyde, 2,3,4-trihydroxy benzaldehyde. Also, it showed LPO inhibitory activity by 68.3% at 6.25 μg/mL. Therefore, we have studied other hydroxy benzaldehydes and their methoxy analogs for LPO, COX-1 and COX-2 enzymes inhibitory activities and found that compound 1 gave the highest COX-2 enzyme inhibitory activity as indicated by a 50% inhibitory concentration (IC(50)) at 9.7 μg/mL. The analogs showed only marginal LPO activity at 6.25 μg/mL. The hydroxy analogs 6, 7 and 9 showed 55%, 61% and 43% of COX-2 inhibition at 100 μg/mL. However, hydroxy benzaldehydes 3 and 12 showed selective COX-1 inhibition while compounds 4 and 10 gave little or no COX-2 enzyme inhibition at 100 μg/mL. At the same concentration, compounds 14, 21 and 22 inhibited COX-1 by 83, 85 and 70%, respectively. Similarly, compounds 18, 19 and 23 inhibited COX-2 by 68%, 72% and 70%, at 100 μg/mL. This is the first report on the isolation of compound 1 from A. leptopus tea with selective COX-2 enzyme and LPO inhibitory activities.

In spite of considerable progress in the methodology for reconstitution of membrane proteins into the liposomes, a successful reconstitution still appears to be more an art than a science. Reconstitution of membrane proteins into bilayers is required for establishing several aspects of the functions of membrane proteins and lipids and for elaborating models of naturally occurring membranes.Cyclooxygenase (COX)-1 and -2 (also prostaglandin endoperoxide H(2) synthase, PGHS-1 and -2) belong to the class of monotopic membrane proteins. Membrane-binding domains of both COX-1 and -2 contain four short, consecutive, amphipathic alpha-helices (A, B, C, and D). Crystal structures of the COXs indicate that basic, hydrophobic, and aromatic residues in the membrane-binding domain are oriented away from the protein core and form a surface on the enzyme, which has been proposed to interact with the lipid bilayer (1).In this chapter, we describe a fast and efficient method for direct incorporation of COX-1 and -2 isozymes - as models for monotopic integral membrane proteins - into preformed liposomes containing fatty acids without loss of activity.

Cherries contain bioactive anthocyanins that are reported to possess antioxidant, anti-inflammatory, anticancer, antidiabetic and antiobese properties. The present study revealed that red sweet cherries contained cyanidin-3-O-rutinoside as major anthocyanin (>95%). The sweet cherry cultivar "Kordia" (aka "Attika") showed the highest cyanidin-3-O-rutinoside content, 185 mg/100 g fresh weight. The red sweet cherries "Regina" and "Skeena" were similar to "Kordia", yielding cyanidin-3-O-rutinoside at 159 and 134 mg/100 g fresh weight, respectively. The yields of cyanidin-3-O-glucosylrutinoside and cyanidin-3-O-rutinoside were 57 and 19 mg/100 g fresh weight in "Balaton" and 21 and 6.2 mg/100 g fresh weight in "Montmorency", respectively, in addition to minor quantities of cyanidin-3-O-glucoside. The water extracts of "Kordia", "Regina", "Glacier" and "Skeena" sweet cherries gave 89, 80, 80 and 70% of lipid peroxidation (LPO) inhibition, whereas extracts of "Balaton" and "Montmorency" were in the range of 38 to 58% at 250 microg/mL. Methanol and ethyl acetate extracts of the yellow sweet cherry "Rainier" containing beta-carotene, ursolic, coumaric, ferulic and cafeic acids inhibited LPO by 78 and 79%, respectively, at 250 microg/mL. In the cyclooxygenase (COX) enzyme inhibitory assay, the red sweet cherry water extracts inhibited the enzymes by 80 to 95% at 250 microg/mL. However, the methanol and ethyl acetate extracts of "Rainier" and "Gold" were the most active against COX-1 and -2 enzymes. Water extracts of "Balaton" and "Montmorency" inhibited COX-1 and -2 enzymes by 84, and 91 and 77, and 87%, respectively, at 250 microg/mL.

We have determined the bioactive constituents in the higher Basidiomycetes edible mushrooms Pholiota nameko, Lentinus edodes, and Pleurotus ostreatus using in vitro lipid peroxidation (LPO) and cyclooxygenase enzyme (COX) inhibitory assays. Ethyl acetate extract of nameko was the most active against LPO, with an inhibition value of 81% at 250 mu g/mL. Shiitake and oyster mushroom extracts inhibited LPO by 39% and 52%, respectively, at 250 mu g/mL. In the COX assay, the ethyl acetate extracts of nameko, shiitake, and oyster mushrooms inhibited COX-1 and -2 enzymes by 39%, 34%, and 54% and 87%, 92%, and 75%, respectively, at 250 mu g/mL. A bioassay-guided purification yielded linoleic acid (1), ergosterol (2), ergosterol peroxide (3), and a glycosphingolipid (4) along with a glyceride fraction (GF), as confirmed by spectroscopic methods. All three mushrooms have shown the presence of compounds 1 and 3 as common constituents. Compound 2 was isolated from both nameko and shiitake mushrooms. Compound 4 was isolated from shiitake and oyster mushrooms. The GF obtained from oyster mushrooms inhibited LPO by 92% and COX-1 and -2 enzymes by 43% and 92%, respectively, at 25 mu g/mL.

The tender shoots of Calamus ornatus, one of the food items consumed by the native people, Kanawan Aytas, in the Bataan region of the Philippines, have not been studied before. A bioassay-guided investigation of its methanolic extract afforded non-nutritive functional agents (NFAs), steroidal saponins 1-3, along with its aglycone (4). The NFAs 1-4 inhibited cyclooxygenase enzymes, COX-1 and -2, by 47%, 43%, 33%, and 53% and 71%, 75%, 78%, and 73%, respectively, at 28.2, 24.2, 21.2 and 60.4μM. Treatment of breast (MCF-7), CNS (SF-268), lung (NCI-H460), colon (HCT-116) and gastric (AGS) cancer cell lines with the extract at 100μg/ml reduced cell proliferation. Similarly, the pure NFAs 2 and 3 reduced the cell viability of breast, CNS, lung, colon and gastric cancer cell lines by 37.5%, 22.4%, 53.3%, 58.2%, 40.3% and 29.8%, 21.3%, 45.6%, 37.1%, 25.0%, respectively, at 24.2 and 21.2μM. The 50% reduction in cell viability (IC50) concentrations of 2 and 3 against these cancer cell lines were 8.8, 6.1, 7.5, 23.8, 12.1 and 3.8, 7.1, 3.3, 14.3, 12.1μM, respectively. This is the first report on the isolation of steroidal saponins from C. ornatus shoots and their antiinflammatory and tumor cell proliferation inhibitory activities. Therefore, our results suggest that the Kanawan Aytas may yield health benefits from rattan-shoots in their diet.