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Polymeric nanoparticles (NPs) represent one of the most promising tools in nanomedicine and have been extensively studied for the delivery of water-insoluble drugs. However, the efficient loading of therapeutic enzymes and proteins in polymer-based nanostructures remains an open challenge. Here, we report a synthesis method for a new enzyme delivery system based on cross-linked enzyme aggregates (CLEAs) encapsulation into poly(lactide- co-glycolide) (PLGA) NPs. We tested the encapsulation strategy on four enzymes currently investigated for enzyme replacement therapy: palmitoyl protein thioesterase 1 (PPT1; defective in NCL1 disease), galactosylceramidase (GALC; defective in globoid cell leukodystrophy), alpha glucosidase (aGLU; defective in Pompe disease), and beta glucosidase (bGLU; defective in Gaucher's disease). We demonstrated that our system allows encapsulation of enzymes with excellent activity retention (usually around 60%), thus leading to functional and targeted nanostructures suitable for enzyme delivery. We then demonstrated that CLEA NPs efficiently deliver PPT1 in cultured cells, with almost complete enzyme release occurring in 48 h. Finally, we demonstrated that enzymatic activity is fully recovered in primary NCL1 fibroblasts upon treatment with PPT1 CLEA NPs.

The stability of enzyme-conjugated magnetic iron oxide nanoparticles in plasma is of great importance for in vivo delivery of the conjugated enzyme. In this study, β-glucosidase was conjugated on aminated magnetic iron oxide nanoparticles using the glutaraldehyde method (β-Glu-MNP), and further PEGylated via N-hydroxysuccinimide chemistry. The PEG-modified, β-glucosidase-immobilized magnetic iron oxide nanoparticles (PEG-β-Glu-MNPs) were characterized by hydrodynamic diameter distribution, zeta potential, Fourier transform infrared spectroscopy, transmission electron microscopy, and a superconducting quantum interference device. The results showed that the multidomain structure and magnetization properties of these nanoparticles were conserved well throughout the synthesis steps, with an expected diameter increase and zeta potential shifts. The Michaelis constant was calculated to evaluate the activity of conjugated β-glucosidase on the magnetic iron oxide nanoparticles, indicating 73.0% and 65.4% of enzyme activity remaining for β-Glu-MNP and PEG-β-Glu-MNP, respectively. Both magnetophoretic mobility analysis and pharmacokinetics showed improved in vitro/in vivo stability of PEG-β-Glu-MNP compared with β-Glu-MNP. In vivo magnetic targeting of PEG-β-Glu-MNP was confirmed by magnetic resonance imaging and electron spin resonance analysis in a mouse model of subcutaneous 9L-glioma. Satisfactory accumulation of PEG-β-Glu-MNP in tumor tissue was successfully achieved, with an iron content of 627±45 nmol Fe/g tissue and β-glucosidase activity of 32.2±8.0 mU/g tissue.

Severe deficiency in lysosomal β-glucuronidase (β-glu) enzymatic activity results in mucopolysaccharidosis (MPS) VII, an orphan disease with symptoms often appearing in early childhood. Symptoms are variable, but many patients have multiple organ disorders including neurological defects. At the cellular level, deficiency in β-glu activity leads to abnormal accumulation of glycosaminoglycans (GAGs), and secondary accumulation of GM2 and GM3 gangliosides, which have been linked to neuroinflammation. There have been encouraging gene transfer studies in the MPS VII mouse brain, but this is the first study attempting the correction of the >200-fold larger and challenging canine MPS VII brain. Here, the efficacy of a helper-dependent (HD) canine adenovirus (CAV-2) vector harboring a human GUSB expression cassette (HD-RIGIE) in the MPS VII dog brain was tested. Vector genomes, β-glu activity, GAG content, lysosome morphology and neuropathology were analyzed and quantified. Our data demonstrated that CAV-2 vectors preferentially transduced neurons and axonal retrograde transport from the injection site to efferent regions was efficient. HD-RIGIE injections, associated with mild and transient immunosuppression, corrected neuropathology in injected and noninjected structures throughout the cerebrum. These data support the clinical evaluation of HD CAV-2 vectors to treat the neurological defects associated with MPS VII and possibly other neuropathic lysosomal storage diseases.

It is unknown whether the bioavailability of isoflavones is affected by the concomitant ingestion of glucosides or aglycones. This study was designed to investigate the effects of soymilk-based beverages containing different types of isoflavones on their absorption, excretion, and metabolism. Twelve healthy volunteers consumed 3 kinds of soymilk: untreated soymilk, beta-glucosidase-treated soymilk, and fermented soymilk. Blood samples were collected after 0, 1, 2, 3, 4, 5, 6, 7, 8, and 24 h. Urine samples were collected from 0 to 48 h. Concentrations of isoflavones and daidzein metabolites in serum and urine were measured by liquid chromatography-mass spectrometry. After the ingestion of soymilk, the total concentration of isoflavones in serum rose slowly and reached a maximum of 0.94 +/- 0.39 micromol/L at 6.0 +/- 1.2 h. However, beta-glucosidase-treated soymilk and fermented soymilk increased the serum isoflavone concentration significantly more quickly with maximum concentrations at 1.0 h of 1.75 +/- 0.33 micromol/L and 2.05 +/- 0.32 micromol/L, respectively. The urinary excretion of isoflavones after ingesting of these aglycone-enriched preparations was significantly greater than after consumption of untreated soymilk up to 8 h after injection, but not thereafter. The total and individual concentrations of isoflavones in serum and urine did not differ when subjects consumed the 2 aglycone-enriched soymilks. However, in equol producers (n = 5), the ingestion of ESM tended to increase urinary excretion of equol compared with the consumption of FSM (P = 0.08). These results demonstrated that the isoflavone aglycones of soymilk were absorbed faster and in greater amounts than their glucosides in healthy adults and that the metabolism of isoflavones might be affected by the type of soymilk consumed.

Background: Olive pollen is an important cause of allergy in Mediterranean countries. More than 50% of olive-pollen-allergic patients are sensitized against the 1,3-beta-glucanase Ole e 9. To date, prophylactic and therapeutic treatments using purified recombinant allergens have not been studied in animal models of olive pollen allergy., Methods: BALB/c mice were immunized against Ole e 9 combining intraperitoneal injections of the allergen in Al(OH)3 with airway allergen challenges. A prophylactic treatment was performed by intranasal administration of a mixture of the recombinant fragments of the allergen prior to Ole e 9 sensitization. Serum levels of specific IgE, IgG1, IgG2a and IgG2b were measured by ELISA, and total IgE levels by sandwich ELISA. Bronchoalveolar lavage and lungs from mice were collected to study airway inflammation by light microscopy., Results: BALB/c mice immunized against Ole e 9 developed a predominantly Th2-like immune response with allergen-specific immunoglobulin induction and airway inflammation accompanied by the infiltration of eosinophils, lymphocytes, and neutrophils in the lung. Prophylactic treatment by intranasal application of the recombinant fragments of Ole e 9 avoids airway inflammation induced by sensitization with this allergen although the levels of Ole e 9-specific antibodies remain unchanged., Conclusions: Prophylactic intranasal treatment with recombinant fragments of Ole e 9 prevents airway inflammation triggered by immunization to this allergen in a murine model of type I allergy., (Copyright (c) 2006 S. Karger AG, Basel.)

Symptomatic patients with Gaucher disease (GD) (acid beta-glucosidase [Gcase] deficiency) are treated with injectable human recombinant GCase. Treatment results in significant decreases in lipid storage in liver, spleen, and bone marrow, but the generalized osteopenia and focal bone lesions present in many adult patients are refractory to treatment. A double-blind, 2-arm, placebo-controlled trial of alendronate (40 mg/d) was performed in adults with GD who had been treated with enzyme for at least 24 months. Primary therapeutic endpoints were improvements in (1) bone mineral density (BMD) and content (BMC) at the lumbar spine, and (2) focal lesions in x-rays of long bones assessed by a blinded reviewer. There were 34 patients with GD type 1 (age range, 18-50 years) receiving enzyme therapy who were randomized for this study. After 18 months, DeltaBMD at the lumbar spine was 0.068 +/- 0.21 and 0.015 +/- 0.034 for alendronate and placebo groups, respectively (P =.001). Long-bone x-rays showed no change in focal lesions or bone deformities in any subject in either arm. Alendronate is a useful adjunctive therapy in combination with enzyme replacement therapy (ERT) for the treatment of GD-related osteopenia in adults, but it cannot be expected to improve focal lesions.

Lysosomal storage diseases (LSDs) are monogenic disorders of metabolism caused by deficiency of hydrolytic enzymes. In several LSDs, cells of the reticuloendothelial (RE) system are the primary targets of the disease. Exogenous administration of recombinant enzymes overproduced in mammalian cells has proved effective for treating the systemic phenotype in nonneuropathic patients with LSDs. However, for the treatment of diseases with primary involvement of the RE system, the production of the therapeutic enzyme in insect cells could be an alternative and advantageous method because glycoproteins expressed in insect cells carry carbohydrates of the pauci-mannose or core-type. These recombinant enzymes are in principle already poised to be internalized by cells that express mannose receptors, including macrophages. Here, we demonstrate that three baculovirus-expressed enzymes, protective protein/cathepsin A (PPCA), neuraminidase (Neu1), and beta-glucosidase, were readily taken up and restored lysosomal function in enzyme-deficient mouse macrophages. The capacity of recombinant PPCA and Neu1 to clear the lysosomal storage in target cells was assessed in PPCA-/- mice, a model of galactosialidosis. Intravenously injected PPCA-/- mice efficiently internalized the corrective enzymes in resident macrophages of many organs. In addition, treated mice showed overall clearance of lysosomal storage in the most affected systemic organs, kidney, liver, and spleen. Our results suggest that ERT with baculovirus-expressed enzymes might be an effective treatment for nonneuropathic patients with galactosialidosis and possibly for others with LSDs that primarily involve the RE system.

1. Effects of preservation method (drying or air-tight storage of whole grain and ensiling of rolled high-moisture grain) and beta-glucanase supplementation (Econase) on apparent ileal amino acid digestibilities and metabolisable energy content of barley were evaluated with Ross broiler chickens. In addition, the effect of barley preservation method was assessed using Leghorn cockerels. 2. Birds were given either a semi-purified soyabean meal basal diet or a mixture of the basal diet and barley (50:50 on dry matter basis). Apparent ileal digestibilities (AID) of nutrients were assessed using the slaughter technique. AID of nutrients and nutrient digestibility measured using excreta (AED) were determined using chromium mordanted straw as an indigestible marker. 3. In broilers, AID of amino acids, dry matter and organic matter were lower for dried than air-tight stored barley, particularly for diets based on ensiled barley. In cockerels, barley preservation method had no effect on amino acid AID. The AED of nutrients and nitrogen corrected apparent metabolisable energy content (AMEn) was highest for ensiled barley across both experiments. 4. beta-glucanase supplementation increased nutrient digestibility, phosphorus retention and AMEn content of air-tight stored and dried barley diets in particular but had only negligible effects on ensiled barley. Beta-glucanase improved the AID of amino acids in dried barley but not in air-tight stored or ensiled barley. 5. Amino acid digestibilities were lower in broilers than cockerels and the effect of barley preservation on feeding value of barley was different for broilers and cockerels.

Activity of supplemental enzymes in a barley-soybean-maize based diet at 60, 75 and 90 degrees C pelleting temperatures was studied using feed viscosity, in-vitro enzyme activity and broiler performance data. High pelleting temperatures increased feed viscosity but supplemented enzymes reduced the viscosity at all three temperatures levels by 11, 14 and 17%, respectively. Water intake and losses in excreta of birds were found to be affected by feed viscosity. Activity of cellulase enzyme, measured using the radial diffusion method, was unaffected at 60 and 75 degrees C, but reduced by 73% in feed processed at 90 degrees C. Enzymes increased the weight gain of broilers by 11.1% at 90 degrees C, but no effect could be seen at low pelleting temperatures possibly due to high dietary protein and energy contents. Feed intake was unaffected by enzymes. Birds consumed 6% more feed and grew 9% faster when the pelleting temperature was increased from 60 to 75 degrees C. Reduced feed intake and daily weight gain observed at 90 degrees C could be fully compensated by the enzyme supplementation. High pelleting temperature reduced energy metabolizability (3.2%) and nitrogen utilization (4%) but enzyme almost compensated them (by 3.3% and 2.6%, respectively). No interaction could be detected between the pelleting temperatures and enzymes. It is concluded that pelleting temperatures as high as 90 degrees C drastically reduce cellulase activity, energy and nitrogen utilization thus lowering broiler performance. Either the remaining activity of cellulase or other thermostable enzymes can prevent the losses.

Over the last decade several attempts have been made to generate an active drug from an inactive precursor, by the action of an enzyme present predominantly at the tumour site. The aim was to develop a new, less cytotoxic strategy for the treatment of cancer, by exploiting properties distinguishing neoplastic and normal cells. In fact, monoclonal antibodies were used to carry enzymes at the tumour sites, in a two-step approach, known as Antibody Directed Enzyme Prodrug Therapy (ADEPT). We reviewed the experimental and clinical considerations of this strategy and we presented its problems and limitations. We concluded that ADEPT holds the potential of an effective and relatively non-toxic treatment of cancer and it is expected that the research which is in progress will make ADEPT an important element of the anticancer armament.

We describe a novel version of antibody-directed enzyme prodrug therapy (ADEPT), with the use of amygdalin as prodrug. Amygdalin is a naturally occurring cyanogenic glycoside, which can be cleaved by sweet almond beta-glucosidase to yield free cyanide. If amygdalin could be activated specifically at the tumour site, then malignant cells would be killed without the systemic toxicity usually associated with chemotherapy. To this end, we have conjugated beta-glucosidase to a tumour-associated monoclonal antibody (MAb) (HMFG1) and the conjugate has been tested in vitro for specificity and cytotoxicity subsequent to activation of amygdalin. Amygdalin was cytotoxic to HT1376 bladder cancer cells only at high concentrations, whereas the combination of amygdalin with HMFG1-beta-glucosidase enhanced the cytotoxic effect of amygdalin by 36-fold. When 2 concentrations of HMFG1-beta-glucosidase were compared, the toxic effect was dose dependent. The cytotoxicity of amygdalin was also enhanced by the MAb-enzyme conjugate even when the unbound conjugate was removed from the medium prior to exposure to amygdalin and the cells were washed. In addition to the cytotoxic effect, we also demonstrated specificity, using a MAb-enzyme conjugate that does not recognise the HT1376 bladder cancer cells. Finally, we studied the cytotoxic effect of the conjugate in co-culture of HMFG1-positive and-negative cell lines (HT 1376 and U87MG cells). We demonstrated that the rate of surviving cells corresponds well to the percentage of U87MG (HMFG1-negative) cells in the flask. Our findings indicate that ADEPT is more effective than non-directed enzyme activation of a prodrug and can result in a non-toxic cancer therapy.

1. Separate balance experiments were conducted to assess the potential of 2 commercial enzyme supplements to improve the nutritive value of dehulled lupin kernels. One supplement (enzyme A) contained primarily xylanase, pentosanase, hemicellulase activities and the other (enzyme B) primarily beta-glucanase, hemicellulase and pectinase activities. 2. The enzymes were added at 0, 0.25, 0.50, 0.75 and 1.00 g/kg in diets containing (g/kg) lupins 300, sorghum 543, casein 91, celite (as marker) 20, and vitamins and minerals 46. Control diets, with and without enzyme supplementation contained sorghum and casein at 800 and 134 g/kg, respectively, and no lupins. 3. Growth rates and food conversion ratios (FCR) of birds over 7 days were not affected by lupin inclusion or enzyme supplementation. FCR of broilers fed on the sorghum diet was improved by enzyme A but not by enzyme B. 4. Ileal starch digestibilities were slightly lower in birds fed on the lupin control diet (no enzyme) compared to the basal control diet. 5. Enzyme A increased the AME of the lupins from 10.01 MJ/kg DM to 11.65 MJ/kg DM when added at 0.5 g/kg. Higher rates of supplementation did not lead to further increases in AME values. 6. Enzyme A did not improve starch digestion in the diets but insoluble non-starch polysaccharides concentration in the digesta decreased (50.41-42.71 g/g acid insoluble ash marker) with increasing enzyme supplementation, suggesting that the improvement in AME was the result of increased fermentation of fibre in the hindgut. 7. Enzyme B did not affect the AME of lupins nor the ileal digestibility of nutrients, but caused an increase in the concentrations of soluble non-starch polysaccharides in the ileal digesta of chickens (19.21-35.77 mg/ml). This was accompanied by an increase in ileal digesta viscosity (11.4-34.2 m.Pa/s).

Two experiments with Leghorn chicks and data from five publications were analyzed to determine whether a simple general equation could be used to predict the relationship between the amount of a crude enzyme added to a diet and chick performance. The maximum improvements in weight gain and feed: gain ratio in Leghorn chicks fed rye diets containing different concentrations of enzymes were as high as 61 and 42%, respectively. Regression analyses demonstrated that there was usually a high linear correlation (r2 > 0.9, P < 0.05) between the concentration of the enzyme when transformed into its logarithmic value and weight gain or the feed:gain ratio. The general prediction equation was Y = A + B(logX), where Y is the performance value (i.e., weight gain, grams), A is the intercept (y-axis), B is the slope of the line (change in performance per units of enzyme in the diet), and X is the amount of enzyme in the diet. The slope of the line provides an index of the overall efficacy of the enzyme treatment. The log-linear model shows that for every ninefold increase in amount of enzyme in the diet (i.e., when the amount was increased to 10 times the starting amount), there was only a doubling of improvement in chick performance. High correlations (r2 values) were also obtained when data from the literature were analyzed. The equation was applicable to different classes and ages of poultry fed diets containing rye, wheat, barley, and lupins. These studies demonstrate that there is a linear relationship between the amount of enzyme added to the diet, when expressed as a logarithmic value, and the corresponding performance of chickens.

Barlican, a beta-glucanase enzyme obtained from Trichoderma reesei, was produced by a fermentation process and subjected to a series of toxicological tests to document its safety for use as a feed additive. The enzyme product was examined for general oral toxicity, inhalation toxicity, irritation to eye and skin, skin sensitization and mutagenic potential. An extensive literature search on the production organism was also conducted. Furthermore, safety for target species was assessed in a 28-day oral toxicity study with broilers. A strong skin-sensitizing potential of the beta-glucanase enzyme was detected, but no other evidence of oral or inhalation toxicity, mutagenic potential, eye or skin irritancy was found. Feeding of the beta-glucanase enzyme at dietary levels up to 10,000 ppm in the 90-day subchronic toxicity study in rats did not induce noticeable signs of toxicity. In addition, no adverse effects were observed when broiler chicks were fed dietary concentrations of the beta-glucanase enzyme up to eight times the daily recommended dose. It is therefore concluded that this beta-glucanase preparation is safe for use in feed of the intended target species. However, some occupational health precautions should be taken to avoid skin contact and inhalation, as is the case for almost all enzyme proteins.

Three hull-less barleys, Washonupana (WSNP), Waxbar (WXB), and Bangsa (BGS), were fed to broiler chicks in 21% protein diets containing 0.5% cholesterol in replicate trials. A corn-based diet, with added cholesterol, served as a control. Alternate diets were supplemented with beta-glucanase (ENZ). beta-glucan content ranged from 4.9% to 6.1% and soluble dietary fiber (SDF) from 3.6% to 7.5% in the barleys. Data from the two trials were pooled for statistical analysis by the SAS General Linear Models procedure. In body weight gain, chicks fed WSNP-ENZ were lower (P less than 0.05) than all other treatments. The beta-glucanase supplement to the WXB and BGS barley tended to improve gains, but the differences were not significant for either barley. Feed to gain ratios were lowest (P less than 0.0001) for corn fed chicks and lower (P less than 0.05 to P less than 0.0001) for those fed the barley + ENZ diets compared to barley -ENZ. Chicks fed barley diets had lower (P less than 0.05) total serum cholesterol (TSC) and LDL-cholesterol than those fed corn diets, regardless of ENZ supplementation. For chicks on barley -ENZ diets, TSC levels for WSNP, WXB, and BGS were 146, 152, and 142 mg/dl respectively and for chicks on barley + ENZ diets, 218, 200, and 178 mg/dl. LDL-cholesterol levels followed the same trend and there was little difference in serum triglycerides. The BGS + ENZ lowered TSC 30% from the corn control compared to 10.7% and 18% for WSNP + ENZ and WXB + ENZ, suggesting additional hypocholesterolemic factors, possibly tocotrienol and SDF other than 1----3, 1----4 beta-D-glucans.