Acute and Subchronic Toxicities and Safety Pharmacology Studies of a Bacillus Subtilisin in Dogs.

Subtilisin NAT, a Bacillus subtilisin, is widely applied as a functional food and considered to be one of the most exploitable potential oral thrombolytic agents. Subtilisin QK, another Bacillus subtilisin, is a serine protease fermented by Bacillus subtilis 02 and has a better thrombolytic effect. Therefore, subtilisin QK is typically used for evaluating the safety of Bacillus subtilisins. Here, we conduct several good laboratory practice (GLP)-compliant studies in non-rodent animal, i.e., in Beagle dogs, including acute toxicity, subchronic toxicity, and safety pharmacology studies. No adverse effects were evident in the acute and 28-d subchronic toxicity studies at doses up to 40000 FU/kg and 16000 FU/kg/d, respectively. In evaluating the pharmacological safety of up to 2000FU/kg subtilisin QK, we found no significant differences between the electrocardiograms, blood pressures, and respiration of beagle dogs. These findings suggest the safety of Bacillus subtilisin, providing reliable pharmacological and toxicological data for its development and popularization as a functional food and drug.

Synthesis of sustained release/controlled release nanoparticles carrying nattokinase and their application in thrombolysis.

Thrombus-related diseases have a high mortality rate and are seriously threatening human life and health. Nattokinase (NK), which has a strong thrombolytic effect, can treat thrombotic diseases. In this study, NK-conjugated magnetite nanoparticles (NK-MNPs) were prepared to accurately deliver NK to the thrombus site. Fe3 O4, carboxymethyl chitosan and sodium alginate were combined to form magnetite nanoparticles (MNPs), which were prepared to encapsulate NK. The mean diameter of NK-MNPs was 168.9±4.8 nm, and the zeta potential was -33.8±0.9 mV. The release percentage reached a plateau in approximately 12 h, with 65.24% NK released. Magnetic targeting experiments showed that the light transmittance of the solution reached 90%. The results from the in vitro thrombolysis experiments demonstrated the sustained release thrombolysis potential of NK-MNPs. A hemolysis experiment demonstrated that the hemolysis rate of NK-MNPs was less than 5% at an enzymatic activity of 50-150 IU/mL. Moreover NK-MNPs were stored for 90 days at 4 °C and still maintained an enzyme activity above 90%. In conclusion, NK-MNPs hold great promise for improved thrombolytic efficacy, with sustained release and magnetic targeting.

Oral administration of alcalase potato protein hydrolysate-APPH attenuates high fat diet-induced cardiac complications via TGF-ß/GSN axis in aging rats.

Consumption of high fat diet (HFD) is associated with increased cardiovascular risk factors among elderly people. Aging and obesity induced-cardiac remodeling includes hypertrophy and fibrosis. Gelsolin (GSN) induces cardiac hypertrophy and TGF-ß, a key cytokine, which induces fibrosis. The relationship between TGF-ß and GSN in aging induced cardiac remodeling is still unknown. We evaluated the expressions of TGF-ß and GSN in HFD fed 22 months old aging SD rats, followed by the administration of either probucol or alcalase potato protein hydrolysate (APPH). Western blotting and Masson trichrome staining showed that APPH (45 and 75 mg/kg/day) and probucol (500 mg/kg/day) treatments significantly reduced the aging and HFD-induced hypertrophy and fibrosis. Echocardiograph showed that the performance of the hearts was improved in APPH, and probucol treated HFD aging rats. Serum from all rats was collected and H9c2 cells were cultured with collected serums separately. The GSN dependent hypertrophy was inhibited with an exogenous TGF-ß in H9c2 cells cultured in HFD+ APPH treated serum. Thus, we propose that along with its role in cardiac fibrosis, TGF-ß also acts as an upstream activator of GSN dependent hypertrophy. Hence, TGF-ß in serum could be a promising therapeutic target for cardiac remodeling in aging and/or obese subjects.

Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease.

Natto, a fermented soybean product, has been consumed as a traditional food in Japan for thousands of years. Nattokinase (NK), a potent blood-clot dissolving protein used for the treatment of cardiovascular diseases, is produced by the bacterium Bacillus subtilis during the fermentation of soybeans to produce Natto. NK has been extensively studied in Japan, Korea, and China. Recently, the fibrinolytic (anti-clotting) capacity of NK has been recognized by Western medicine. The National Science Foundation in the United States has investigated and evaluated the safety of NK. NK is currently undergoing a clinical trial study (Phase II) in the USA for atherothrombotic prevention. Multiple NK genes have been cloned, characterized, and produced in various expression system studies. Recombinant technology represents a promising approach for the production of NK with high purity for its use in antithrombotic applications. This review covers the history, benefit, safety, and production of NK. Opportunities for utilizing plant systems for the large-scale production of NK, or for the production of edible plants that can be used to provide oral delivery of NK without extraction and purification are also discussed.

Design, purification and assessment of GRP78 binding peptide-linked Subunit A of Subtilase cytotoxic for targeting cancer cells.

BACKGROUND: Targeted therapies for cancer, especially the malignant cancer, are always restricted by the deficiency of tumor-specific drug delivery methods. Subtilase cytotoxic is a virulent cytotoxin, and the subunit A (SubA) of it is able to destroy the structure of glucose-regulated protein 78 (GRP78) to induce cell apoptosis, and to be expected as anti-cancer drugs, however, the ubiquitous receptor of subunit B of Subtilase cytotoxic (SubB) restricts its application on cancer therapy. RESULTS: The present study constructed and expressed a fusion protein of GBP-SubA in E. coli Rosetta (DE3) system, in which the subunit B of Subtilase cytotoxic was replaced by GRP78 binding peptide (GBP). The fusion protein was expressed in inclusion body form. Subsequently, the denaturation/renaturation process and Ni-column purification were performed. Our data indicated the purified GBP-SubA could bind GRP78 existed on cancer cell surface specifically, internalize into cells to inactivate intracellular GRP78 and induce apoptosis. Moreover, the apoptosis induction effect of GBP-SubA was enhanced obviously along with the increased cancer cell surface GBP78. CONCLUSIONS: It indicates that the recombinant GBP-SubA possesses the dual functions of GBP and SubA to induce cancer cell apoptosis specifically, revealing that GBP-SubA holds important implications for developing as an anti-cancer peptide drug. A schematic representation of the construction and function of GBP-SubA.

A pilot study on the serum pharmacokinetics of nattokinase in humans following a single, oral, daily dose.

CONTEXT: Nattokinase is a serine protease and is derived from natto, a traditional Japanese, fermented, soybean food meal. Multiple authors have described the significant fibrinolytic, antithrombotic, and antihypertensive effects of natto. Nattokinase has been growing in popularity for use as a dietary supplement for the benefit of cardiovascular health. Little is known regarding the pharmacokinetic and pharmacodynamic properties of this enzyme, and the bioavailability of nattokinase is currently unknown. OBJECTIVE: This study intended to (1) detect nattokinase directly and immunologically, (2) show that nattokinase and/or its metabolites were detectable in human blood following ingestion of a commercial preparation, and (3) chart a pharmacokinetic dosing effect for nattokinase. DESIGN: The research team designed the pilot study as an in vivo, human clinical trial. Healthy human subjects responded to an advertisement and were screened. Subjects who satisfied both inclusion and exclusion criteria were enrolled into the study. Subjects were then instructed to orally ingest a single capsule containing a known concentration of nattokinase immediately following a baseline blood draw. Subsequent blood draws occurred over a 24-h period. SETTING: This study was conducted in Oakland, California, at a clinical reference laboratory and was performed with the approval of an institutional review board (IRB) to ensure that appropriate ethical standards were met. PARTICIPANTS: Eleven healthy participants (five male, six female, ages 21-65), who met eligibility criteria, were enrolled. INTERVENTION(S): Administration of nattokinase occurred orally with the ingestion of a single daily dose (2000 FU) of nattokinase. Capsules, each containing approximately 100 mg of nattokinase, in softgel form (NSK-SD, Japan Bio Science Laboratory, Osaka, Japan), were used in the study. OUTCOME MEASURE(S): Baseline blood samples were collected, and participants were observed swallowing a single capsule of the nattokinase supplement before returning at 2, 4, 8, 12, 24, and 48 h post ingestion for subsequent blood draws. The presence of nattokinase in serum was measured by an enzyme-linked immunosorbent assay (ELISA), using a rabbit, polyclonal, antinattokinase-capture antibody. A pharmacokinetic pattern was observed for nattokinase between baseline and 48 h postdose. RESULTS: Peak serum levels of nattokinase were observed at approximately 13.3 h ± 2.5 h (mean ± standard error) postdose. Statistically significant increases in binding were detectable from baseline when comparing averaged data at time points t = 2 h-t = 24 h. CONCLUSIONS: These results provided the first evidence that nattokinase can be measured directly in the blood of humans. The results further suggest that a larger, more extensive, pharmacokinetic study of nattokinase is warranted. Additionally, looking for intact enzyme and bioactive nattokinase peptides should be a consideration for future studies investigating the pharmacokinetic profile of nattokinase.

A subcytotoxic dose of subtilase cytotoxin prevents lipopolysaccharide-induced inflammatory responses, depending on its capacity to induce the unfolded protein response.

Subtilase cytotoxin (SubAB) is the prototype of a newly identified family of AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli. SubAB specifically cleaves the essential endoplasmic reticulum (ER) chaperone BiP (GRP78), resulting in the activation of ER stress-induced unfolded protein response (UPR). We have recently shown that the UPR following ER stress can suppress cellular responses to inflammatory stimuli during the later phase, in association with inhibition of NF-kappaB activation. These findings prompted us to hypothesize that SubAB, as a selective UPR inducer, might have beneficial effects on inflammation-associated pathology via a UPR-dependent inhibition of NF-kappaB activation. The pretreatment of a mouse macrophage cell line, RAW264.7, with a subcytotoxic dose of SubAB-triggered UPR and inhibited LPS-induced MCP-1 and TNF-alpha production associated with inhibition of NF-kappaB activation. SubA(A272)B, a SubAB active site mutant that cannot induce UPR, did not show such effects. In addition, pretreatment with a sublethal dose of SubAB, but not SubA(A272)B, protected the mice from LPS-induced endotoxic lethality associated with reduced serum MCP-1 and TNF-alpha levels and also prevented the development of experimental arthritis induced by LPS in mice. Collectively, although SubAB has been identified originally as a toxin associated with the pathogenesis of hemolytic uremic syndrome, the unique ability of SubAB to selectively induce the UPR may have the potential to prevent LPS-associated inflammatory pathology under subcytotoxic conditions.

Antihypertensive effects of continuous oral administration of nattokinase and its fragments in spontaneously hypertensive rats.

To determine whether the antihypertensive effect of nattokinase is associated with the protease activity of this enzyme, we compared nattokinase with the fragments derived from nattokinase, which possessed no protease activity, in terms of the effect on hypertension in spontaneously hypertensive rats (SHR). In the continuous oral administration test, the groups were given a basic diet alone (control), the basic diet containing nattokinase (0.2, 2.6 mg/g diet) or the basic diet containing the fragments derived from nattokinase (0.2, 0.6 mg/g diet). The group fed the basic diet containing high-dosage nattokinase (2.6 mg/g diet) showed significant reductions in systolic blood pressure (SBP), diastolic blood pressure (DBP) and plasma fibrinogen level, compared with control group and no influence on activities of renin and angiotensin-converting enzyme (ACE, EC 3.4.15.1), and plasma angiotensin II level in the renin-angiotensin system. The treatment of the basic diet containing high-dosage fragments (0.6 mg/g diet) significantly decreased SBP, DBP and plasma angiotensin II level in plasma but the treatment did not influence on plasma fibrinogen level. These results suggest that nattokinase and its fragments are different from each other in the mechanism to reduce hypertension. Nattokinase, retained its protease activity after absorbance across the intestines, may decrease blood pressure through cleavage of fibrinogen in plasma. The fragments, which absorbed as nattokinase-degradation products, prevents the elevation of plasma angiotensin II level to suppress hypertension.

Purification and Characterization of a Thrombolytic Enzyme Produced by a New Strain of Bacillus subtil.

Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48°C and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.

[Evaluating thrombolytic efficacy and thrombus targetability of RGDS-liposomes encapsulating subtilisin FS33 in vivo].

A novel fibrinolytic enzyme subtilisin FS33, which exhibits much higher activity for decomposing fibrin than urokinase, was purified from Douchi, a traditional soybean-fermented food in China. In order to increase bio-utilization and thrombus targetability of subtilisin FS33 labeled by fluorescein isothiocyanate (FITC), the surface modified liposomes encapsulating subtilisin FS33 and FITC with a synthetic peptide Arg-Gly-Asp-Ser (RGDS), being putatively a specific antagonist of fibrinogen receptor on platelet membrane, were prepared and used to evaluate the therapeutic efficacy in a rat model thrombotic carotid artery. The arterial thrombosis was induced by applying two pieces of filter paper (1 x 2 cm) saturated with 10% of ferric chloride (FeCl3). The rats were infused via the jugular vein with either liposomes carrying BSA (control group) or RGDS-liposomes carrying subtilisin FS33 at doses of 2000 and 4000 U/kg. The plasma of the group infused with RGDS-liposomes showed higher antithrombotic and fibrinolytic activity than did the control group within 15-120 min after infusing. The higher the dose was gived, the higher the activity was shown. APTT(activiated partial thromboplastin time), PT (prothrombin time) and TT (thrombin time) were extended remarkably (P < 0.05, P < 0.01), and FDP (fibrinogen degradation products) also increased greatly (P < 0.01), while ELT (euglobulinlysis time) decreased obviously (P < 0.05). FITC content in heart and brain evidently increased (P < 0.05), and results of D-dimer test were all positive. In addition, the venous thrombi in brain and kidney were dissolved totally or partly as observed by patholgical section. All these indicated that subtilisin FS33 enhanced the antithrombotic and fibrinolytic activities in rat, and RGDS-liposomes improved, in a certain degree, the thrombolytic specificity for targeting to thrombus.

Development of a nattokinase-polysialic acid complex for advanced tumor treatment.

Cancer-associated thrombus (CAT) impedes delivery of nanoparticles to tumor sites and also inhibits the ability of immune cells to detect and attack these tumors, particularly in advanced tumors with old thrombi. Nattokinase (NK) is an extract from a popular Japanese food, natto, which consists of boiled soybeans fermented with bacteria. Nattokinase exerts strong fibrinolytic and thrombolytic activities and can unblock blood vessels. To deliver NK to thrombus sites in tumors, we modified the surface of NK with polysialic acid (PSA), which formed complexes via electrostatic interactions, resulting in NK-PSA. Particle size and zeta potential of NK-PSA were evaluated, and differential scanning calorimetry, Fourier-transform infrared spectroscopy, and morphological analyses of NK-PSA were performed. To determine the efficacy of the NK-PSA complex on delivery of nanoparticulate drugs, sialic acid-modified doxorubicin liposomes (DOX-SAL) were used as a model drug. In vivo pharmacokinetic and tissue distribution analyses showed that the blood clearance rate of DOX-SAL was significantly enhanced by NK-PSA, and NK-PSA increased accumulation of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) labeled SAL (DiR-SAL) in tumors. Analysis of anti-tumor efficacy showed that the combination of NK-PSA and DOX-SAL enhanced anti-tumor activity. These results suggested that NK-PSA combined with DOX-SAL may be an effective strategy to clear CAT and increase the ability of nanoparticles and immune cells to reach tumors.

The effects of nattokinase supplementation on collagen-epinephrine closure time, prothrombin time and activated partial thromboplastin time in nondiabetic and hypercholesterolemic subjects.

The aim of this study was to investigate whether supplementation with nattokinase, which is considered one of the most active functional ingredients found in natto, alters hemostatic factors. Subjects presenting with hypercholesterolemia (serum cholesterol: 200-280 mg dL-1) were randomly divided into nattokinase and placebo groups (n = 50, respectively). No significant between-group differences were found at baseline in collagen-epinephrine closure time (C-EPI CT), prothrombin time (PT), or activated partial thromboplastin time (aPTT). After 8 weeks of treatment, the nattokinase group exhibited significant increases in C-EPI CT, PT, and aPTT. The nattokinase group showed significantly greater increases in C-EPI CT (P = 0.001) and aPTT (P = 0.016) than the placebo group. Moreover, at 8 weeks, the nattokinase group showed a significantly higher C-EPI CT than the placebo group (P = 0.001). Additionally, a significant correlation between PT and aPTT was observed (r = 0.491, P < 0.001). In conclusion, nattokinase supplementation was associated with prolonged C-EPI CT and aPTT in nondiabetic and borderline-to-moderate hypercholesterolemic subjects.

Effects of nattokinase on blood pressure: a randomized, controlled trial.

The objective of this study was to examine the effects of nattokinase supplementation on blood pressure in subjects with pre-hypertension or stage 1 hypertension. In a randomized, double-blind, placebo-controlled trial, 86 participants ranging from 20 to 80 years of age with an initial untreated systolic blood pressure (SBP) of 130 to 159 mmHg received nattokinase (2,000 FU/capsule) or a placebo capsule for 8 weeks. Seventy-three subjects completed the protocol. Compared with the control group, the net changes in SBP and diastolic blood pressure (DBP) were -5.55 mmHg (95% confidence interval [CI], -10.5 to -0.57 mmHg; p<0.05) and -2.84 mmHg (CI, -5.33 to -0.33 mmHg; p<0.05), respectively, after the 8-week intervention. The corresponding net change in renin activity was -1.17 ng/mL/h for the nattokinase group compared with the control group (p<0.05). In conclusion, nattokinase supplementation resulted in a reduction in SBP and DBP. These findings suggest that increased intake of nattokinase may play an important role in preventing and treating hypertension.

Pharmacotherapy of Vitreomacular Traction.

Vitreomacular traction occurs due to incomplete or anomalous posterior vitreous detachment. Over time, the vitreous pulls anteriorly and causes retinal distortion and eventually reduced vision. Traditionally, vitreomacular traction was treated with vitrectomy surgery. In the past few years, there is a paradigm shift towards pharmacologic vitreolysis, which involves the intravitreal injection of enzymatic and non-enzymatic agents that facilitate posterior vitreous detachment. Many agents have been investigated and trialled including plasmin, microplasmin (Ocriplasmin), hyaluronidase, nattokinase, chondroitinase and dispase. This review will focus on the progress and current status in this research.

Multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer: Design, synthesis, and dissolving thrombus.

Thrombotic events affect many individuals in a number of ways, all of which can cause significant morbidity and mortality. Nattokinase (NK), as a novel thrombolytic drug, has been used for thrombolytic therapy. It not only possesses plasminogen activator activity, but also directly digests fibrin through limited proteolysis. However, it may undergo inactivation and denaturation in the harsh external environment. In this study, a multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer was fabricated and used as a carrier for NK protection and delivery. Different arm numbers of polyethylene glycol-polyglutamic acid peptide dendrimers (x-PEG(G3 )x , x = 2, 4, 6, 8) were designed, prepared, and characterized by 1 H NMR and FTIR. Then, x-PEG(G3 )x were loaded with NK to form nanocomposites. Their size and morphology were determined by dynamic light scattering and transmission electron microscopy. Enzyme activity was evaluated via UV-Vis absorbance spectra, fluorescence spectra, circular dichroism spectra, and zeta potential measurements. The study reveals that the obtained x-PEG(G3 )x /NK nanocomposites possess high enzyme activity. In addition, the nanocomposites show increased viability of rat macrophage cells, and excellent thrombolysis ability in vitro and in vivo. This work establishes a multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer with potential application in NK carrier and thrombolytic therapy. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1687-1696, 2018.

Development of surface-engineered PLGA nanoparticulate-delivery system of Tet1-conjugated nattokinase enzyme for inhibition of Aß40 plaques in Alzheimer's disease.

According to the World Health Organization, globally there are around 18 million patients suffering from Alzheimer's disease (AD), and this number is expected to double by 2025. The pathophysiology of AD includes selective deposition of Aß peptide in the mitochondria of cells, which inhibits uptake of glucose by neurons and key enzyme functions. Current drug treatments for AD are unable to rectify the underlying pathology of the disease; they only provide short-term symptomatic relief, so there is a need for the development of newer treatment regimes. The antiamyloid activity, antifibrinolytic activity, and antithrombotic activity of nattokinase holds potential for the treatment of AD. As nattokinase is a protein, its stability restricts its usage to a greater extent, but this limitation can be overcome by nanoencapsulation. In this work, we successfully synthesized polymeric nanoparticles of nattokinase and characterized its use by different techniques: transmission electron microscopy, scanning electron microscopy, DTS Nano, differential scanning calorimetry, Fourier-transform infrared spectroscopy, thioflavin T-binding assay, in vitro drug release, antifibrinolytic activity, and in vivo antiamyloid activity. As brain targeting of hydrophilic drugs is complicated due to the stringent nature of blood-brain barrier, in the current experimental study, we conjugated poly(lactic-co-glycolic acid) (PLGA)-encapsulated nattokinase with Tet1 peptide, which exhibits retrograde transportation properties because of its affinity to neurons. Our study suggests that PLGA-encapsulated nattokinase polymeric nanoparticles are able to downregulate amyloid aggregation and exhibit antifibrinolytic activity. The encapsulation of nattokinase in PLGA did not affect its enzyme activity, so the prepared nanoformulation containing nattokinase can be used as an effective drug treatment against AD.

Toxicological assessment of nattokinase derived from Bacillus subtilis var. natto.

Subtilisin NAT, commonly known as "nattokinase," is a fibrinolytic enzyme produced by the bacterial strain B. subtilis var. natto, which plays a central role in the fermentation of soybeans into the popular Japanese food natto. Recent studies have reported on the potential anticoagulatory and antihypertensive effects of nattokinase administration in humans, with no indication of adverse effects. To evaluate the safety of nattokinase in a more comprehensive manner, several GLP-compliant studies in rodents and human volunteers have been conducted with the enzyme product, NSK-SD (Japan Bio Science Laboratory Co., Ltd., Japan). Nattokinase was non-mutagenic and non-clastogenic in vitro, and no adverse effects were observed in 28-day and 90-day subchronic toxicity studies conducted in Sprague-Dawley rats at doses up to 167 mg/kg-day and 1000 mg/kg-day, respectively. Mice inoculated with 7.55 × 10(8) CFU of the enzyme-producing bacterial strain showed no signs of toxicity or residual tissue concentrations of viable bacteria. Additionally consumption of 10 mg/kg-day nattokinase for 4 weeks was well tolerated in healthy human volunteers. These findings suggest that the oral consumption of nattokinase is of low toxicological concern. The 90-day oral subchronic NOAEL for nattokinase in male and female Sprague-Dawley rats is 1000 mg/kg-day, the highest dose tested.

Low dose of GRP78-targeting subtilase cytotoxin improves the efficacy of photodynamic therapy in vivo.

Photodynamic therapy (PDT) exerts direct cytotoxic effects on tumor cells, destroys tumor blood and lymphatic vessels and induces local inflammation. Although PDT triggers the release of immunogenic antigens from tumor cells, the degree of immune stimulation is regimen-dependent. The highest immunogenicity is achieved at sub-lethal doses, which at the same time trigger cytoprotective responses, that include increased expression of glucose-regulated protein 78 (GRP78). To mitigate the cytoprotective effects of GRP78 and preserve the immunoregulatory activity of PDT, we investigated the in vivo efficacy of PDT in combination with EGF-SubA cytotoxin that was shown to potentiate in vitro PDT cytotoxicity by inactivating GRP78. Treatment of immunocompetent BALB/c mice with EGF-SubA improved the efficacy of PDT but only when mice were treated with a dose of EGF-SubA that exerted less pronounced effects on the number of T and B lymphocytes as well as dendritic cells in mouse spleens. The observed antitumor effects were critically dependent on CD8+ T cells and were completely abrogated in immunodeficient SCID mice. All these results suggest that GRP78 targeting improves in vivo PDT efficacy provided intact T-cell immune system.

Downstream Processing, Formulation Development and Antithrombotic Evaluation of Microbial Nattokinase.

The present research work describes the downstreaming of nattokinase (NK) produced by Bacillus subtilis under solid state fermentation; and the role of efficient oral formulation of purified NK in the management of thrombotic disorders. Molecular weight of purified NK was estimated to be 28 kDa with specific activity of 504.4 FU/mg. Acid stable nattokinase loaded chitosan nanoparticles (sNLCN) were fabricated for oral delivery of this enzyme. Box-Behnken design (BBD) was employed to investigate and validate the effect of process (independent) variables on the quality attributes (dependent variables) of nanoparticles. The integrity, conformational stability and preservation of fibrinolytic activity of NK (in both free and sNLCN forms) were established by SDS-PAGE, CD analysis and in vitro clot lytic examination, respectively. A 'tail thrombosis model' demonstrated significant decrease in frequency of thrombosis in Wistar rats upon peroral administration of sNLCN in comparison with negative control and free NK group. Furthermore, coagulation analysis, namely the measurement of prothrombin and activated partial thromboplastin time illustrated that sNLCN showed significantly (p < 0.001) higher anti-thrombotic potential in comparison to the free NK. Further, sNLCN showed anti-thrombotic profile similar to warfarin. This study signifies the potential of sNLCN in oral delivery of NK for the management of thrombotic disorders.

A single-dose of oral nattokinase potentiates thrombolysis and anti-coagulation profiles.

Our aim was to determine the quantitative effects of a single-dose of Nattokinase (NK) administration on coagulation/fibrinolysis parameters comprehensively in healthy male subjects. A double-blind, placebo-controlled cross-over NK intervention study was carried out in 12 healthy young males. Following the baseline blood draw, each subject was randomized to receive either a single-dose of 2,000 FU NK (NSK-SD, Japan Bio Science Laboratory Co., Ltd) or placebo with subsequent cross-over of the groups. Subjects donated blood samples at 2, 4, 6 and 8 hours following administration for analysis of coagulation/fibrinolysis parameters. As a result, D-dimer concentrations at 6, and 8 hours, and blood fibrin/fibrinogen degradation products at 4 hours after NK administration elevated significantly (p < 0.05, respectively). Factor VIII activity declined at 4 and 6 hours (p < 0.05, respectively), blood antithrombin concentration was higher at 2 and 4 hours (p < 0.05, respectively), and the activated partial thromboplastin time prolonged significantly at 2 and 4 hours following NK administration (p < 0.05 and p < 0.01, respectively). All the changes, however, were within the normal range. In conclusion, thus, a single-dose of NK administration appears enhancing fibrinolysis and anti-coagulation via several different pathways simultaneously.