HA Removal Research Articles

Humic acid removal using cellulose acetate membranes grafted with poly (methyl methacrylate) and aminated using tetraethylenepentamine

Graft copolymerization of cellulose acetate (CA) and poly (methyl methacrylate) (PMMA) was synthesized through free radical polymerization in the presence of cerium sulfate (CS) as initiator under nitrogen atmosphere in an aqueous solution. During the grafting reactions, the effects of polymerization time and temperature on the grafting were investigated. Furthermore, functionalization of the synthesized product was done using amine group (tetraethylenepentamine, TEPA). The results from Nuclear Magnetic Resonance (1H NMR) spectra confirmed a successful grafting of PMMA on the CA membrane surfaces. Zeta potential (ζ), field emission scanning electron microscopy (FESEM), and atomic absorption spectrophotometer (AAS) characterization studies were done. The maximum removal efficiencies for un-grafted CA (un-g-CA), CA-g-PMMA, and CA-g-PMMA-TEPA membranes at pH of 7.0 were 34.5%, 83.3%, and 99.1%, respectively. The removal percentage results were detected to increase with increasing in the regeneration cycles. At the end of the fourth cycle, the HA removal percentages were 41.6%, 87.4%, and 99.9% for un-g-CA, CA-g-PMMA and CA-g-PMMA-TEPA membranes, respectively.

Abstract 641: PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances tumor infiltrating CD8+ T cell accumulation and improves checkpoint inhibitor efficacy in murine syngeneic breast cancer models

Abstract Hyaluronan (HA) is an extracellular glycosaminoglycan that accumulates in the tumor microenvironment (TME) of many solid tumors and is associated with rapid tumor progression and poor prognosis. In preclinical studies, enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) remodels the TME, reduces tumor interstitial fluid pressure, decompresses tumor blood vessels, and facilitates delivery of chemotherapeutics. We have previously shown that PEGPH20-mediated HA degradation enhances anti-PD-L1 and anti-PD1 efficacy in an HA accumulating murine pancreatic tumor model (Rosengren, AACR 2016 poster #4886). Accordingly, we aimed to extend these checkpoint blockade studies into orthotopic breast cancer models; while further elucidating the effect of PEGPH20-mediated HA degradation on modulating tumor infiltrating lymphocytes (TILs). The mammary fat pads of BALB/c mice were inoculated with either EMT-6 cells or 4T1/HAS3, a 4T1 cell line engineered to over-express hyaluronan synthase-3. During tumor progression, both EMT-6 and 4T1/HAS3 mammary carcinomas accumulate high levels of HA (average 730 ng/mg and 1408 ng/mg, respectively). When treated with PEGPH20 alone (0.0375mg/kg), EMT-6 tumor growth was inhibited by ≥30% (p=0.01). Additional EMT-6 studies were conducted to evaluate PEGPH20 in combination with anti-PD-L1. PEGPH20 increased anti-PD-L1 efficacy by 38% relative to anti-PD-L1 alone (86% vs 62.4% tumor growth inhibition (TGI), p=0.0024). Comparable tumor growth experiments were conducted in the 4T1/HAS3 model. TILs were evaluated by flow cytometry. PEGPH20-mediated HA removal enhanced both checkpoint efficacy and CD8+ T cell recruitment. Specifically, PEGPH20 alone (1mg/kg) increased anti-PD-L1 efficacy by 411% relative to anti-PD-L1 alone (93% vs 18.2% TGI, p<0.0001) and increased the accumulation of CD8+ TIL by 176% (p=0.0025). Taken together, the data suggest that tumor HA accumulation may act as a barrier to immune cell access and negatively modulate CD8+ TILs, and that PEGPH20-mediated HA reduction facilitates increased access of CD8+ T cells. This increased recruitment may contribute to the enhanced anti-PD-L1 efficacy observed when combined with PEGPH20. A Phase 1 trial is ongoing to evaluate PEGPH20 plus checkpoint blockade in patients with advanced or metastatic non-small cell lung cancer and in patients with locally advanced or metastatic gastric adenocarcinoma (NCT02563548). Citation Format: Renee Clift, Jisook Lee, Curtis B. Thompson, Yujun Huang. PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances tumor infiltrating CD8+ T cell accumulation and improves checkpoint inhibitor efficacy in murine syngeneic breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 641. doi:10.1158/1538-7445.AM2017-641

Flocculation performance of cationic polyacrylamide with high cationic degree in humic acid synthetic water treatment and effect of kaolin particles

Abstract In current research, the flocculation performance of cationic polyacrylamide flocculant PAMC with high cationic degree was investigated in HA synthetic water treatment, and the effect of kaolin particles was studied in kaolin-HA flocculation. Influencing factors, such as pH, PAMC dosage, initial HA concentration, cationic degree, stirring speed and stirring time, were investigated and optimized for HA removal efficiency. Two-dimensional fractal dimension of the flocs was analyzed based on flocs images, and FTIR analysis was accomplished to understand chemical structures of the flocs. Three-dimensional excitation emission matrix spectra was measured to analyze the changes in organic composition and concentraion. Finally, the HA and kaolin-HA synthetic water flocculation mechanism were studied and summarized based on analysis of zeta potential and pH. The results showed that 80.7% and 91.2% of HA removal efficiency was achieved in HA and kaolin-HA flocculation process by using 12.5 mg/L and 4–6 mg/L of PAMC, respectively. Moreover, partial colloidal HA was absorbed by kaolin particles before flocculation and was removed in flocculation through synergetic effect from kaolin particles. Charge neutralization and bridging effect was confirmed to be dominant in acidic and alkaline environment, respectively.

Enhanced efficiency in HA removal by electrocoagulation through optimizing flocs properties: Role of current density and pH

Abstract The performance of electrocoagulation (EC) significantly depends on flocs properties that could be improved through regulating electrochemical parameters. However, little information has been collected to date regarding the relationship between flocs characteristics and electrochemical parameters. In this study, the influences of pH and current density ( j ) were investigated to characterize the flocs generated from EC process with aluminum anode. The results indicated that higher j performed better on HA removal due to a larger Al dosage. A highest efficiency in HA removal by EC process occurred at weak acidic range, where the flocs presented a stronger adsorption ability. Higher j and higher water pH led to a larger flocs size. The EC time to form the largest flocs was consistent with that to reach zero point charge, where surface charge favors the aggregation of Al(OH) 3 precursors to form large flocs. Flocs strength was positively correlated with applied j and negatively correlated with water pH. The flocs generated with low applied j and at low initial pH had a higher value of BET surface area and a more porous surface structure.

Adsorption of Soil-Derived Humic Acid by Seven Clay Minerals: A Systematic Study

AbstractHumic acid (HA)-clay complexes are well known for their contribution to soil structure and environmental processes. Despite extensive research, the mechanisms governing HA adsorption are yet to be resolved. A systematic study was conducted to characterize the adsorption of a soil-derived HA to seven clay minerals. Clay surfaces affected HA adsorption directly due to structural differences and indirectly by altering solution pH. The following order of HA removal was obtained for the clay minerals at their natural pH: illite ≫ palygorskite > kaolinite > sepiolite > montmorillonite = hectorite ≫ talc. Removal of HA (precipitation and adsorption) by kaolinite and illite was attributed to the low pH they induce, resulting in protonation of the clay and HA surfaces. In spite of the low pH, the zeta potential for HA remained negative, which promoted HA adsorption to the protonated clay surfaces by ligand exchange. Ionic strength did not affect HA adsorption to clay minerals with low zeta potentials, indicating that charge screening is not a major mechanism of HA adsorption for these minerals, and supporting the suggestion that ligand exchange is the main adsorption mechanism to pH-dependent sites. The increase in ionic strength did, however, promote HA adsorption to clay minerals with high zeta potentials. At pH 8–9 the order of HA affinity for clay minerals was: palygorskite >>sepiolite > montmorillonite = hectorite > kaolinite > illite > talc, emphasizing strong HA interactions with the fibrous clays. This strong affinity was attributed to their large surface areas and to strong interactions with OH groups on these clay surfaces. Results indicated that HA did not enter the intracrystalline channels of the fibrous clays but suggested that their macro-fiber structure facilitates HA adsorption. The sorption of HA to kaolinite further increased in the presence of Cu2+, and the sorption of Cu2+ increased in the presence of HA, due to a number of synergistic effects. This study emphasizes the diverse effects of clay structure and solution chemistry on HA adsorption.

Abstract A46: Hyaluronan-dependent growth of human triple negative breast cancer MDA-MB-468 in a mouse xenograft model with HA-high stroma

Abstract Hyaluronan (also known as hyaluronic acid or HA) is a megadalton sized glycosaminoglycan present in the extracellular matrix. The abnormal accumulation of HA in the solid tumor microenvironment (TME) of several human cancers, including pancreatic, breast, colon and prostate, has been well studied and is associated with poor prognosis. In certain breast cancer specimens with a HA-high status, HA is found to be significantly associated with the stromal compartment rather than with tumor cells. To understand the interaction between tumor cells and a HA containing stromal compartment, we engineered a HA-high stromal cell line by lentiviral transduction of the human HAS3 gene into Balb/c 3T3 fibroblast cells. The 3T3/HAS3 cells produced significant amounts of HA in vitro, which was shown to bind to the HA-low/CD44-high breast cancer cell line MDA-MB-468 at the cell surface. When co-grafted with MDA-MB-468 cells in mice, 3T3/HAS3 cells promoted the in vivo growth of MDA-MB-468 cells. Immunohistochemical analysis of tumor xenograft samples showed that MDA-MB-468 cells were surrounded by HA-high stromal cells, closely resembling the tumor morphology observed in human breast cancer specimens. Tumor growth in this co-grafting model was highly dependent on the presence of 3T3/HAS3 cells, and required HA produced by 3T3/HAS3 cells. Ganciclovir blocked the growth of MDA-MB-468 co-inoculated with 3T3/HAS3/hsv-TK, as did HA removal either by the expression of the HA degrading enzyme PH20, directly in 3T3/HAS3 cells or by exogenous administration of PEGPH20, a pegylated version of PH20 engineered for extended half-life. Interestingly, the presence of 3T3/HAS3 cells was found to be most critical during early stages of tumor progression and was non-essential for tumor maintenance once tumors were fully established, as ganciclovir had little impact on tumor growth when tumor size was larger than 500 mm3 but prevented tumor growth when administered at inoculation and inhibited tumor growth when treatment started at a smaller tumor size (200 mm3, 90.4% TGI). We have developed a tumor xenograft model that mimics breast cancer with a HA-high status in the tumor stromal compartment. Further characterization of this model will provide insight into the mechanisms by which increased levels of HA and the associated changes in the TME promote disease progression in breast cancer. Citation Format: Chunmei Zhao, Mathieu Marella, Lei Huang, Anne Kultti, Susan Zimmerman, Caroline EN Chou, Jesse Bahn, Adrian Radi, Zhongdong Huang, H Michael Shepard, Christopher D. Thanos. Hyaluronan-dependent growth of human triple negative breast cancer MDA-MB-468 in a mouse xenograft model with HA-high stroma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A46.

Abstract 2463: PEGPH20 increases the anticancer activity of standard chemotherapy combinations, vincristine (VIN) and D actinomycin (DACT), in a Wilms’ xenograft model

Abstract Hyaluronan (HA), which accumulates in the tumor microenvironment of many solid tumors, is associated with tumor progression and negative clinical outcomes. Preclinical studies have demonstrated that PEGPH20-mediated HA removal from HA-rich xenograft tumors in mice decreases tumor interstitial fluid pressure and water content, resulting in decompression of tumor vasculature, increased tumor perfusion and enhanced chemotherapeutic activity. Accordingly, the HA degrading enzyme, pegylated recombinant human hyaluronidase PH20 (PEGPH20), is currently being evaluated in clinical trials with selected treatment regimens in several malignancies, including metastatic pancreatic adenocarcinoma, gastric cancer, breast cancer and non-small cell lung cancer. Advances in treatment for pediatric Wilms’ tumor patients have significantly increased survival, but salvage chemotherapy for relapsed pediatric patients remains challenging. To evaluate the role of HA in this disease, 19 Wilms’ tumor histological specimens were surveyed for HA status using a novel probe (Jadin 2014). 79% (15/19) of the samples stained strongly for HA when compared to non-tumor kidney tissue, prompting additional preclinical studies. In brief, a human Wilms’ tumor cell line, WT-CLS1, was transduced with hyaluronan synthase-3 (HAS3). The subsequent WT-CLS1/HAS3 cells produced more HA and WT-CLS1/HAS3 xenograft tumors grew significantly faster than parental WT-CLS1 tumors. To evaluate the efficacy of PEGPH20 in combination with Wilms’ tumor chemotherapy (CTX) combinations, WT-CLS1/HAS3 cells were inoculated adjacent to the tibial periosteum of nude mice and tumor growth was monitored via ultrasonography. When tumors reached ∼250 mm3, mice were staged into treatment groups: vehicle control, PEGPH20, vincristine (VIN) plus Dactinomycin (DACT), and PEGPH20+VIN+DACT at different dose/frequency combinations. Average tumor growth inhibition (TGI) and overall survival for PEGPH20+VIN+DACT-treated mice were superior to TGI and survival of VIN+DACT treatment alone. In separate studies WT-CLS1/HAS3 tumor-bearing mice were staged into three groups: vehicle control, low dose PEGPH20 (0.0375 mg/kg, iv) and high dose PEGPH20 (1 mg/kg, iv). Animals were administered the hypoxyprobe pimonidazole (60 mg/kg, ip) three hours prior to sacrifice, and whole tumors were removed and processed via immunofluorescence. High dose PEGPH20 reduced this measure of tumor hypoxia by 5% (p = 0.023). Taken together, these data demonstrate that HA accumulation in Wilms’ tumor is common, and treatment with PEGPH20 hyaluronidase can increase CTX efficacy and reduce tumor hypoxia in a preclinical model of this disease. These results support further studies with PEGPH20 in combination with chemotherapy in preclinical models of Wilms’ tumor and suggest investigation in this pediatric patient population may be warranted. Citation Format: Jessica Cowell, Susan J. Zimmerman, Mathieu Marrela, Ping Jiang, Peter J. Houghton, Michael J. LaBarre, Daniel C. Maneval, Curtis B. Thompson, Xiaoming Li. PEGPH20 increases the anticancer activity of standard chemotherapy combinations, vincristine (VIN) and D actinomycin (DACT), in a Wilms’ xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2463.

Effects of sodium, magnesium, and calcium salts on the coagulation performance of cucurbit [8]uril for humic acid removal from synthetic seawater

This study evaluates the coagulation performance of cucurbit [8]uril (CB [8]) for HA removal and the effects of salts on this process. Experimental results showed that HA removal efficiency improved as the increase of CB [8] dosage. When the CB [8] dosage was 0.2mmol/L, less than 50% of the HA was removed from the synthetic freshwater at pH above 7.5. The presence of salts improved both the solubility and coagulation capacity of CB [8], which varied depending on the type of anion and cation, respectively. For the synthetic seawater with 1.0mmol/L of Ca2+, 8.0mmol/L of Mg2+, and 0.4mol/L of Na+ co-existing, the HA removal efficiency increased considerably from less than 50% to 83–90% at pH7.5–8.5. Relative to the amount of HA removed by adsorption, an additional 20–40% of the HA was removed by CB [8] coagulation under the same water quality condition. Further in-depth analysis showed that HA coagulation by CB [8] is primarily a physical process. In addition to charge neutralization and electrical double-layer compression, the formation of hydrogen bonds between CB [8] and HA also contributed to HA destabilization and led to a complete loss in stability before all the negative charges on the HA were neutralized.

Abstract P1-03-09: Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances efficacy of eribulin mesylate (HALAVEN®) in triple negative breast cancer xenografts

Abstract Hyaluronan (hyaluronic acid, HA), a glycosaminoglycan found in tissue throughout the body, overaccumulates in the tumor microenvironment (TME) of many non-hematologic malignancies, including breast cancer. HA overaccumulation in breast cancer patients correlates with tumor progression and decreased survival (Tammi 2008). Pegylated recombinant human hyaluronidase PH20 (PEGPH20), an investigational therapeutic agent entering Phase 3 clinical development in pancreatic cancer, enzymatically removes HA from the TME. In preclinical animal models, PEGPH20-mediated HA degradation is associated with remodeling of the tumor stroma, reduction of tumor pressure, expansion of tumor blood vessels and facilitated delivery of chemotherapy (Thompson 2010, Provenzano 2012, Jacobetz 2013). Accordingly, preclinical studies investigated the combination of PEGPH20 with eribulin mesylate (ERI, HALAVEN®), a microtubule dynamics inhibitor with a novel mechanism of action (Towle 2001, Jordan 2005), currently approved for treatment of certain patients with advanced breast cancer. NCr nu/nu mice were inoculated subcutaneously with human triple-negative breast cancer (TNBC) HCC1806 or HCC1806/HAS3 cells; the latter subline was engineered to accumulate high HA levels, confirmed by immunohistochemistry, via overexpression of hyaluronan synthase 3 (HAS3). When tumors reached ∼350 mm3, animals were randomly assigned to four treatments groups: vehicle, ERI (0.7 mg/kg, IV, QW), PEGPH20 (37.5 µg/kg, IV, BIW), or ERI plus PEGPH20. In the parental HCC1806 model, addition of PEGPH20 did not significantly change the antitumor effects of ERI. In contrast, combining PEGPH20 with ERI in the HCC1806/HAS3 model increased the antitumor effects of ERI by 27% (94.5% vs. 119.7% TGI, ERI alone vs. ERI+PEGPH20, respectively; p=0.05) and resulted in 6 of 7 complete tumor regressions. In a complementary study in HCC1806/HAS3 tumors evaluating ERI pharmacokinetics with and without PEGPH20, mice were assigned to three treatments groups: ERI (0.5 mg/kg, IV), simultaneous ERI plus PEGPH20 (37.5 µg/kg, IV); or ERI plus PEGPH20 predosed 24 h prior to ERI. Animals were sacrificed at 0.5, 1, 4, 24, 48, 72 and 96 h post ERI dose, and ERI levels in tumor, muscle, plasma and liver were subsequently analyzed by liquid/liquid extraction and LC-MS/MS chromatography. Simultaneous administration of ERI and PEGPH20 increased ERI maximum tumor concentration (Cmax) slightly and approximately doubled ERI tumor exposure (AUC); whereas the 24 h pretreatment with PEGPH20 approximately doubled ERI Cmax and increased ERI AUC more than two-fold. No significant differences in plasma ERI levels were observed between groups, and no significant differences in ERI levels in liver or muscle tissue were observed between groups. Taken together, these data suggest that PEGPH20-mediated HA removal significantly increases both ERI tumor concentrations and antitumor effectiveness in an HA-high TNBC model. A clinical phase 1b/2 clinical trial is planned to evaluate PEGPH20 plus ERI in first-line HER2-negative metastatic breast cancer. Citation Format: Bahn JD, DesJardins C, Condon KB, Fathallah A, Zimmerman S, Maneval DC, Littlefield BA, Thompson CB. Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances efficacy of eribulin mesylate (HALAVEN®) in triple negative breast cancer xenografts. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-03-09.

Preparation and characterization of low fouling novel hybrid ultrafiltration membranes based on the blends of GO−TiO2 nanocomposite and polysulfone for humic acid removal

In this work, graphene oxide (GO)−TiO2 nanocomposite was synthesized by in situ sol−gel reaction at pH=2 using GO nanosheets suspension and titanium isopropoxide precursor. The synthesized GO−TiO2 nanocomposite was explored as a filler to fabricate improved antifouling novel hybrid ultrafiltration membranes for removal of humic acid from aqueous solution. Membranes were fabricated from polymer blend solutions containing polysulfone and GO−TiO2 with varied loading amount (0–5wt%) by the non-solvent induced phase separation (NIPS) method. Contact angle, atomic force microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy and outer surface zeta potential studies were conducted in order to characterise the membranes in terms of roughness, structure, surface properties and charge. The porous hydrophilic hybrid membranes were shown to have an asymmetric structure with improved surface roughness. The water permeability and antifouling capacity of hybrid membranes with 10ppm HA solution were dependent on the loading amount of GO–TiO2. Incorporation of GO–TiO2 nanocomposite was found to improve the antifouling characteristics of the membranes when challenged with HA solutions. Irreversible HA fouling was substantially reduced with increased loading of GO−TiO2 nanocomposite (wt%). The lowest irreversible fouling ratio (3.2%) was obtained for the membrane containing 5wt% nanocomposite (to total wt% of PSf, MG−5). Ultrafiltration of HA solutions of varied concentrations using hybrid membranes was studied at pH=7 and 1bar feed pressure. The removal efficiency of hybrid membranes for HA was controlled by the membrane surface charge concentration, porosity and HA exclusion. The membrane MG−5 had the highest HA removal efficiency for 10ppm HA solution at pH=7.

Synergistic Removal of Humic Acid in Water by Coupling Adsorption and Photocatalytic Degradation Using TiO2/Coconut Shell Powder Composite

The feasibility of applying nanoscale TiO2/coconut shell powder (TCNSP) composite to remove HA in aqueous solution was evaluated, and the optimization of the photocatalytic systems using newly developed TCNSP composite was performed. The developed TCNSP composite has high specific surface area (i.e., 454 m2/g) and great porosity (i.e., 66.9%) with pore size of less than 5 μm. High removal efficiencies (≥95%) of HA were observed due to the significant synergistic effects by coupling adsorption and photocatalytic reaction of TCNSP composite. As the initial concentration of HA increased, the degradation rate (Kapp) decreased due to HA sorption saturation to the surface of TCNSP composite and the photon interception by HA molecules in aqueous solution. Since the increased loading amount of TCNSP composite enhanced the number of active sites,Kappvalues increased until the optimum loading amount of TCNSP composite. As pH values increased, HA removal efficiency decreased due to increasing electrostatic repulsion between HA and TCNSP composite. Based on the response surface methodology, higher HA removal efficiencies were obtained with acidic condition, longer reaction time, and appropriated loading amount of TCNSP. Further pilot-scale study is in progress using TCNSP composite combined with UVC to remove HA from large amounts of surface water (i.e., 200 m3/d).

Salmonella-Based Therapy Targeting Indoleamine 2,3-Dioxygenase Coupled with Enzymatic Depletion of Tumor Hyaluronan Induces Complete Regression of Aggressive Pancreatic Tumors.

Bacterial-based therapies are emerging as effective cancer treatments and hold promise for refractory neoplasms, such as pancreatic ductal adenocarcinoma (PDAC), which has not shown significant improvement in therapy for more than 25 years. Using a novel combination of shIDO-ST, a Salmonella-based therapy targeting the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO), with an enzyme, PEGPH20, which depletes extracellular matrix hyaluronan, we observed extended survival with frequent total regression of autochthonous and orthotopic PDAC tumors. This observation was associated with migration and accumulation of activated polymorphonuclear neutrophils (PMN) from spleens into tumors, which was not seen using a scrambled control (shScr-ST). Purified splenic PMNs from PEGPH20/shIDO-ST-treated mice exhibited significant IDO knockdown and were able to kill tumor targets ex vivo through mechanisms involving FasL and serine proteases. In addition, CD8(+) T cells were observed to contribute to late control of pancreatic tumors. Collectively, our data demonstrate that entry of shIDO-ST and PMNs into otherwise impermeable desmoplastic tumors is facilitated by PEGPH20-mediated HA removal, further highlighting an important component of effective treatment for PDAC.

Abstract 2547: PEGPH20 enhances chemotherapy in patient-derived and traditional cell-derived xenograft NSCLC models

Abstract Hyaluronan (HA) accumulates in the tumor microenvironment (TME) of many solid tumors, including prostate, colon, breast, stomach, ovary, pancreas and lung, and HA accumulation is associated with tumor progression and a negative clinical outcome. Accordingly, the HA degrading enzyme pegylated recombinant human hyaluronidase PH20 (PEGPH20) was developed to allow systemic therapy and enable treatment of HA-high tumors. Preclinical studies have demonstrated that PEGPH20-mediated HA removal from HA-rich xenograft tumors in mice decreases tumor interstitial fluid pressure and water content; resulting in decompression of tumor vasculature, increased tumor perfusion and enhanced chemotherapeutic (CTX) activity. As non-small cell lung cancer (NSCLC) is the most common cancer worldwide and is characterized by high levels of HA (∼30%), we aimed to evaluate PEGPH20 enhancement of anti-tumor activity of NSCLC CTXs. Specifically, using both cell derived xenograft (CDX) models, peritibial H2170/HAS3 human NSCLC xenografts, and patient derived xenograft (PDX) models, we investigated CTX tumor growth inhibition (TGI)+/- PEGPH20. Nude mice were inoculated with either H2170/HAS3 cells adjacent to the tibial periosteum or patient derived tumor fragments of human NSCLCs implanted subcutaneously, and tumor growth was monitored via ultrasonography or caliper, respectively. When tumors reached ∼400 mm3, mice were staged into four groups: (1) vehicle; (2) PEGPH20, (3) CTX(s), or (4) PEGPH20 + CTX(s). PEGPH20 alone depleted tumor HA in both CDX and PDX models. In CDX studies, PEGPH20 increased tumor perfusion, reduced hypoxia and decreased HIF-1α expression (p<0.05). A summary table of the TGI data is shown below. The average TGI for PEGPH20 plus CTX(s) was superior to all CTX(s) alone, suggesting that enzymatic depletion of TME HA increases tumor sensitivity to NSCLC CTXs, likely due to increased CTX access into the TME. PEGPH20 clinical trials in NSCLC are currently in development. Summary of TGIsGroup (n≥5/group)CDX%TGI w/ docetaxelCDX%TGI w/ nab-paclitaxel + carboplatinPDX%TGI w/ docetaxelPDX%TGI w/ gemcitabine + cisplatin(p-value vs. V)Vehicle (V)----PEGPH20 (P)28.5 (p<0.05)11.5 (p>0.05)44.4 (p<0.01)29.3 (p>0.05)CTX(s)19.8 (p>0.05)68.6 (p<0.0001)52.5 (p<0.001)38.4 (p>0.05)P + CTX56.1 (p<0.001)93.5 (p<0.0001)>100 (p<0.0001)76.6 (p<0.01) Citation Format: Jessica A. Cowell, Xiaoming Li, Ping Jiang, Susan Zimmerman, Rebecca Symons, H. Michael Shepard, Daniel C. Maneval, Curtis B. Thompson. PEGPH20 enhances chemotherapy in patient-derived and traditional cell-derived xenograft NSCLC models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2547. doi:10.1158/1538-7445.AM2015-2547

Abstract B86: Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances nab-paclitaxel plus gemcitabine efficacy in human pancreatic cancer xenografts

Abstract Hyaluronan (HA) over-accumulation in the extracellular matrix (ECM) of many solid tumors is associated with tumor progression and poor prognosis. Accordingly, an HA-degrading enzyme, PEGPH20, is being developed to deplete tumor-associated HA in the ECM. In preclinical animal models, enzymatic removal of ECM HA with PEGPH20 is associated with remodeling of the tumor stroma, reduction of tumor interstitial fluid pressure, expansion of tumor blood vessels and facilitated delivery of chemotherapy. As pancreatic ductal adenocarcinoma (PDA) has been identified as a cancer type that accumulates high levels of HA, and the combination of albumin-bound paclitaxel (nab-paclitaxel, NAB) and gemcitabine (GEM) has been shown to improve survival in patients with metastatic pancreatic cancer (Von Hoff 2013), preclinical studies were conducted using human PDA xenograft models to investigate whether PEGPH20 enhanced the anti-tumor activity of NAB and GEM. In brief, nude mice were inoculated with human BxPC3 PDA cells, or with HA-high BxPC3/HAS3 cells, a cell line engineered to over-express HA, adjacent to the right tibial periosteum. Tumor growth was monitored via ultrasonography. When tumors reached ~350 mm3, mice were staged into 8 treatment groups: (1) vehicle control; (2) PEGPH20 alone, 4.5 mg/kg; (3) NAB alone, 10 mg/kg; (4) GEM alone, 180 mg/kg; (5) NAB plus PEGPH20; (6) GEM plus PEGPH20; (7) NAB plus GEM; and (8) NAB plus GEM plus PEGPH20. Vehicle, PEGPH20 and/or NAB was administered intravenously starting on study day 0, and then dosed twice weekly for four weeks. GEM was administered intraperitoneally starting on study day 1 and then dosed weekly for three weeks. Histology of tumors confirmed HA removal in all PEGPH20 treated groups. In the parental BxPC3 model, the addition of PEGPH20 increased the anti-tumor efficacy of NAB plus GEM by 15% (81% vs. 66%, respectively) and extended median survival time (MST) by >31% (68d vs. 52d, respectively); whereas in the HA-high BxPC3/HAS3 model, PEGPH20 increased the anti-tumor efficacy of NAB plus GEM by 34% (104% vs. 70%) and extended MST by >125% (72d vs. 32d, respectively). As Jacobetz et al. (2013) have shown that PEGPH20 treatment increases the intratumoral delivery of GEM in genetically engineered mouse PDA models; we evaluated whether PEGPH20 also improves intratumoral accumulation of paclitaxel in the BxPC3/HAS3 model vs. NAB alone. The co-administration of PEGPH20 with NAB increased intratumoral paclitaxel accumulation by >43% one hour following a single administration. Further, PEGPH20 enhanced NAB plus GEM mediated reduction in the PDA serum biomarkers CA19-9 and CEA in both models. As KRAS mutations occur in 90% of non neuro-endocrine pancreatic tumors (Thomas 2007), and BxPC3 cells are KRAS WT, we repeated these studies in KRAS mutant/HA-low AsPC1 xenografts, and engineered KRAS mutant/HA-high AsPC1/HAS3 PDA xenografts. Mice were inoculated and tumor growth was monitored as described above. Animals were then staged into 8 groups and then dosed when tumors reached ~350 mm3. The PEGPH20 dose in these studies was reduced to 37.5 μg/kg to approximate dosing in ongoing clinical trials (NCT01839487). As predicted by tumor HA level, the addition of PEGPH20 did not increase the anti-tumor efficacy or survival in the HA-low parental AsPC1 model. In the HA-high AsPC1/HAS3 model, the addition of PEGPH20 increased the anti-tumor efficacy of NAB plus GEM by 25% (78% vs. 53%, respectively) and extended MST by >31% (74d vs. 48d, respectively). Taken together, the data suggest that PEGPH20-mediated HA removal significantly increases the anti-tumor efficacy of NAB plus GEM in HA-high mouse models of PDA. Clinical trials are currently ongoing to evaluate PEGPH20 plus NAB plus GEM in stage IV patients with metastatic pancreatic cancer (NCT01839487). Citation Format: Ryan J. Osgood, James F. Skipper, Jessica A. Cowell, Yanling Chen, Li Zhu, Michael E. Bledsoe, Susan J. Zimmerman, David W. Kang, H. Michael Shepard, Daniel C. Maneval, Curtis B. Thompson. Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances nab-paclitaxel plus gemcitabine efficacy in human pancreatic cancer xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B86.

Effect of pH with different purified aluminum species on coagulation performance and membrane fouling in coagulation/ultrafiltration process

The influences of solution pH on coagulation/ultrafiltration (C–UF) process were investigated by using three purified Al species of polyaluminium chloride (PACl). A series of online-simulation experiments were developed to assess the coagulation removal efficiencies (turbidity, UV254), floc properties and membrane fouling in this paper. The results showed that change of pH had a significant impact on coagulation efficiencies, floc properties, membrane flux as well as the whole process. Under acidic condition, the hydrolysis action of aluminum salts was restrained which is bad for charge neutralization. While under alkaline region, absorption was the dominant mechanism to combine HA–Kaolin. Meanwhile, HA is apt to soluble by deprotonating under alkaline region which is hard to remove. These common effects made the experiment results complex. HA removal efficiency of Ala and Alb were higher than that of Alc, but the turbidity removal by Alc was slightly higher under the same pH condition. Flocs generated by Ala at pH 6 had advantages such as larger size and the most loosely structure which contributed the most to alleviating membrane fouling. Membrane fouling with Alb and Alc in alkaline range was more serious than that in acidic range.

Effects of epichlorohydrin–dimethylamine on coagulation and membrane performance of ferric chloride in coagulation–ultrafiltration hybrid process

Cationic polymer epichlorohydrin–dimethylamine (DAM–ECH) was applied as coagulation aid of ferric chloride (FC) in treatment of humic acid–kaolin (HA–Kaolin) simulated water in this study. Impacts of DAM–ECH on coagulation performance, flocs characteristics and membrane fouling of FC in coagulation–ultrafiltration (C–UF) process were investigated under different pH conditions. Results showed that the removal rates of UV254 (ultraviolet adsorption at 254nm) and DOC (dissolved organic carbon) of FC/DAM–ECH was much better than that of FC at same dosage. The optimum coagulation condition was FC at 10mg/L with FC/DAM–ECH mass ratio (MR) of 3:1 based on the consideration of treatment cost and performance. Moreover, turbidity, UV254 and DOC removal rates increased when pH increased from 4 to 6 and then decreased as pH grew from 7 to 10. Thus, the optimum pH for coagulation was also determined to be 6 accordingly. Addition of DAM–ECH also increased sizes of produced flocs and made them looser in structure comparing with FC–HA flocs. Meanwhile, results obtained from ultrafiltration experiments showed that membrane fouling would be reduced by introducing DAM–ECH in coagulation treatment; the optimum membrane performance was given by FC/DAM–ECH under pH 6. In conclusion, proper dose of FC/DAM–ECH under pH 6 could ensure an ideal HA removal rate and meanwhile increase operating duration of C–UF system by restricting membrane fouling.

Optimization of the Coagulation Process for Kaolin and Humic Acid Removal Using Polymeric Aluminum Ferric Sulfate

In the paper, the optimization of the coagulation process for Kaolin and humic acid removal using polymeric aluminum ferric sulfate (PAFS) was studied. In order to obtain the maximum turbidity and humic acid removal efficiency of Kaolin and humic acid simulated wastewater, the optimum coagulation conditions was investigated with the factors of mixing speed and time. Furthermore, mixing speed and time including parameters affecting the coagulation performance such as rapid mixing speed, rapid mixing time, slow mixing speed and slow mixing time using single factor and orthogonal array L9 (34) analysis were examined. The results showed that the optimum single factor of mixing speed and time indicated rapid mixing speed of 350 rpm, rapid mixing time of 1.0 min, slow mixing speed of 60 rpm and slow mixing time of 20 min. Then the orthogonal optimization experiment of mixing speed and time indicated maximum HA removal efficiency was 97.5% at rapid mixing speed of 350 rpm, rapid mixing time of 1.25 min, slow mixing speed of 60 rpm, and slow mixing time of 20 min.

Coagulation and Flotation Conditions of Humic Acid by Dissolved Air Flotation

Abstract Coagulation, flocculation, and dissolved air flotation (DAF) experiments were performed with humic acid to evaluate the influence of operational conditions on removal efficiencies. We investigated coagulation, flocculation, and flotation conditions of humic acid removal using a laboratory-scale DAF system. This paper deals with coagulant type (aluminum sulfate and PSO-M) and the most relevant operational conditions (velocity gradients for coagulation and flocculation, retention time and recycle ratio and flotation time). Results showed that optimal conditions for removing humic acid, yielding C HA removal efficiencies of approximately 85 %, are a recycle ratio of 40 %, coagulant dosages of 0.15 – 0.20 gAl/gHA as aluminum sulfate and 0.03 – 0.12 gAl/gHA as PSO-M, coagulation(400s -1 and 60s), flocculation(60s -1 and 900s or more), and flotation(490 kPa or more and at least 10 min). Key Words : Humic acid, Coagulation, Flotation, Velocity gradient, Kinetic model, DAF 1) 1. Introduction

The disinfection by-products precursors removal efficiency and the subsequent effects on chlorine decay for humic acid synthetic water treated by coagulation process and coagulation–ultrafiltration process

The disinfection by-products (DBPs) precursors removal efficiency and the subsequent effects on chlorine decay of coagulation–ultrafiltration process were studied in a laboratory in comparison with coagulation process. And the relation between flocs properties and the performance of coagulation–ultrafiltration process was investigated by the experiments of flocs size, fractal dimension and the surface charge. The removal efficiency of coagulation–ultrafiltration process showed that turbidity and UV254 could be removed efficiently regardless of polyferric chloride (PFC) dosage and solution pH. Even when dosage was 3mgL−1, the turbidity and UV254 removal efficiencies were higher than 90% in coagulation–ultrafiltration process. The chlorine decay showed that under various technological conditions, the difference between chlorine decay curves obtained by coagulation–ultrafiltration process was less than that obtained by coagulation process. According to the analysis by AQUASIM software, the concentration of fast reducing agents (FRA) in coagulation–ultrafiltration process is lower than that in coagulation process, but the concentration of Slow Reducing Agents (SRA) in coagulation–ultrafiltration process was higher than in coagulation process except dosage=18mgL−1. Comparing the flocs properties to the performance of coagulation–ultrafiltration process, it can be found that when flocs size was small (<200μm), the HA removal efficiency increased with flocs size increasing, while when flocs size was large (>200μm), the HA removal efficiency was influenced mainly by flocs structure. The more compact the flocs, the higher the HA removal efficiency.

Humic Acid Removal from Aqueous Environments by Electrocoagulation Process Using Iron Electrodes

At present study the performance of electrocoagulation process using iron electrodes sacrificial anode has been investigated for removal of HA from artificial aqueous solution. The experiments were performed in a bipolar batch reactor with four iron electrode connected in parallel. Several working parameters, such as initial pH (3, 5, 7, and 9), electrical conductivity (50 V) and reaction time were studied in an attempt to achieve the highest removal capacity. Solutions of HA with concentration equal 20 mg L-1 were prepared. To follow the progress of the treatment, samples of 10 ml were taken at 15, 30, 45, 60, and 75 min interval. Finally HA concentration was measured by UV absorbance at 254 nm (UV254) and TOC concentration was measured by TOC Analyser. The maximum efficiency of HA removal which was obtained in voltage of 50 V, reaction time of 75 min, initial concentration 20 mg L-1, conductivity 3000 µS/Cm and pH 5, is equal to 92.69%. But for natural water samples at the same optimum condition removal efficiency was low (68.8 %). It can be concluded that the electrocoagulation process has the potential to be utilized for cost‐effective removal of HA from aqueous environments.