Quantitative pharmacokinetic evaluation of Subtilisin QK-2 after a bolus IV injection in a rat model using a novel sandwich enzyme-linked immunosorbent assay.

Intravascular thrombosis is a main cause of multiple cardiovascular diseases. A high thrombolytic activity of the microbial fibrinolytic enzyme Subtilisin QK-2, which is highly homologous to Nattokinase, shows great exploitable potential in thrombolytic therapy. However, the lack of a sensitive detection method limits the further analysis of Subtilisin QK-2 in vivo. We prepared a polyclonal antibody and four monoclonal antibodies (IgG1, titers of 1:500,000) to establish a sensitive sandwich ELISA for Subtilisin QK-2 detection. The limit of detection (LOD) of this ELISA was 1.160 ng/mL. The linear range of the standard curve was 1.96-250 ng/mL (R2 = 0.9912). The cut-off value was 0.236. Subsequently, a pharmacokinetic dose (IV bolus) was administered and analyzed with the established ELISA. The concentration-time profiles were best fitted to a two-compartment model. T1/2α values for doses of 2 mg/kg, 4 mg/kg, and 8 mg/kg were 29.90 ±â€¯10.02 min, 27.17 ±â€¯1.96 min, and 21.83 ±â€¯9.95 min, and T1/2ß values were 144.43 ±â€¯49.73 min, 173.46 ±â€¯52.58 min, and 159.49 ±â€¯48.75 min, respectively. Subtilisin QK-2 was eliminated through a mechanism with first-order kinetics. In conclusion, this study provides useful data for further research and clinical applications of Subtilisin QK-2 in the treatment of cardiovascular diseases.

Pharmaceutically modified subtilisins withstand acidic conditions and effectively degrade gluten in vivo.

Detoxification of gluten immunogenic epitopes is a promising strategy for the treatment of celiac disease. Our previous studies have shown that these epitopes can be degraded in vitro by subtilisin enzymes derived from Rothia mucilaginosa, a natural microbial colonizer of the oral cavity. The challenge is that the enzyme is not optimally active under acidic conditions as encountered in the stomach. We therefore aimed to protect and maintain subtilisin-A enzyme activity by exploring two pharmaceutical modification techniques: PEGylation and Polylactic glycolic acid (PLGA) microencapsulation. PEGylation of subtilisin-A (Sub-A) was performed by attaching methoxypolyethylene glycol (mPEG, 5 kDa). The PEGylation protected subtilisin-A from autolysis at neutral pH. The PEGylated Sub-A (Sub-A-mPEG) was further encapsulated by PLGA. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gluten immunogenic epitopes were decreased by 60% in the stomach of mice fed with chow containing Sub-A-mPEG-PLGA (0.2 mg Sub-A/g chow) (n = 9) compared to 31.9% in mice fed with chow containing unmodified Sub-A (n = 9). These results show that the developed pharmaceutical modification can protect Sub-A from auto-digestion as well as from acid inactivation, thus rendering the enzyme more effective for applications in vivo.

Effects of exogenous proteases without or with carbohydrases on nutrient digestibility and disappearance of non-starch polysaccharides in broiler chickens.

The objective of the current study was to evaluate the effect of a subtilisin protease, without or with inclusion of carbohydrases, on digestibility and retention of energy and protein, as well as the solubilization and disappearance of non-starch polysaccharides (NSP) from corn-soybean meal based diets fed to broiler chickens. Two hundred eighty-eight Ross 308 male broiler chickens were used for the experiment. On d 14, the birds were weighed and allocated to 6 treatments and 8 replicates per treatment with 6 birds per replicate. Treatments were: 1) corn-soybean meal based control diet; 2) control diet plus supplemental protease at 5,000 (P5000) protease units (PU)/kg); 3) control plus 10,000 PU/kg protease (P10000); or control plus an enzyme combination containing xylanase, amylase, and protease (XAP) added to achieve protease activity of: 4) 2,500 PU/kg (XAP2500); 5) 5,000 PU/kg (XAP5000); or 6) 10,000 PU/kg (XAP10000). The enzymes in XAP were combined at fixed ratios of 10:1:25 of xylanase:amylase:protease. Data were analyzed by ANOVA and specific orthogonal contrasts between treatments were performed. Addition of xylanase and amylase increased (P < 0.05) the ileal digestibility of protein by 4.2% and 2.1% at XAP5000 and XAP10000, respectively (relative to P5000 and P10000, respectively), exhibiting a plateau after the XAP5000 dose. Increment (P < 0.05) in AME due to protease was evident, particularly in P10000. At the ileal level, XAP reduced (P < 0.05) the flow of insoluble xylose and arabinose, which indicates an increase in the solubilization of arabinoxylan polymers in the small intestine. Protease on its own reduced (P < 0.05) the flow of insoluble arabinose but did not affect the flow of insoluble xylose. XAP reduced (P < 0.05) the pre-cecal flow of insoluble and total glucose and galactose. It was concluded that whereas protease by itself improved nutrient utilization and increased solubilization of NSP components, at the lower dose, a combination of xylanase, amylase, and protease produced effects greater than those of protease alone.

[Experience with clinical administration of new drug Thrombovasim® in vascular surgery].

Recent years have witnessed an increased worldwide interest in medical use of unique naturally occurring enzymes - subtilisins possessing pronounced fibrinolytic and anti-inflammatory properties. The article deals with experience in clinical administration in vascular surgery of new therapeutic agent Thrombovasim® containing pegylated subtilisin as an active substance. Thrombovasim® has a favourable profile of safety, good tolerance and causes no severe haemorrhagic complications. The pharmacological action of Thrombovasim® consists in combination of the targeted effect on the fibrin carcass of the thrombus without participation of the own system of haemostasis and anti-inflammatory effect. In the Russian Federation Thrombovasim® has been used for 6 years predominantly in vascular pathology of lower limbs accompanied by the development of chronic venous insufficiency.

Assessment of the respiratory sensitization potential of proteins using an enhanced mouse intranasal test (MINT).

There remains a need for a simple and predictive animal model to identify potential respiratory sensitizers. The mouse intranasal test (MINT) was developed to assess the relative allergic potential of detergent enzymes, however, the experimental endpoints were limited to evaluation of antibody levels. The present study was designed to evaluate additional endpoints (serum and allergic antibody levels, pulmonary inflammation and airway hyperresponsiveness (AHR)) to determine their value in improving the predictive accuracy of the MINT. BDF1 mice were intranasally instilled on days 1, 3, 10, 17 and 24 with subtilisin, ovalbumin, betalactoglobulin, mouse serum albumin or keyhole limpet hemocyanin; challenged with aerosolized methacholine or the sensitizing protein on day 29 to assess AHR, and sacrificed on day 29 or 30. Under the conditions of this study, evaluation of AHR did not improve the predictive power of this experimental model. Allergic antibody responses and IgG isotype characterization proved to be the most sensitive and reliable indicators of the protein allergenic potential with BAL responses providing additional insight. These data highlight that the evaluation of the respiratory sensitization potential of proteins can be best informed when multiple parameters are evaluated and that further improvements and refinements of the assay are necessary.

Protein micro and nanoencapsulation within glycol-chitosan/Ca²+/alginate matrix by spray drying.

Encapsulation of therapeutic peptides and proteins into polymeric micro and nanoparticulates has been proposed as a strategy to overcome limitations to oral protein administration. Particles having diameter less than 5 µm are able to be taken up by the M cells of Peyer's patches found in intestinal mucosa. Current formulation methodologies involve organic solvents and several time consuming steps. In this study, spray drying was investigated to produce protein loaded micro/nanoparticles, as it offers the potential for single step operation, producing dry active-loaded particles within the micro to nano-range. Spherical, smooth surfaced particles were produced from alginate/protein feed solutions. The effect of operational parameters on particle properties such as recovery, residual activity and particle size was studied using subtilisin as model protein. Particle recovery depended on the inlet temperature of the drying air, and mean particle size ranged from 2.2 to 4.5 µm, affected by the feed rate and the alginate concentration in the feed solution. Increase in alginate:protein ratio increased protein stability. Presence of 0.2 g trehalose/g particle increased the residual activity up to 90%. Glycol-chitosan-Ca(2+)alginate particles were produced in a single step operation, with resulting mean diameter of 3.5 µm. Particles showed fluorescein isothiocyanate labeled bovine serum albumin (BSA)-protein entrapment with increasing concentration toward the particle surface. Similar, limited release profiles of BSA, subtilisin and lysozyme were observed in gastric simulation, with ultimate full release of the proteins in gastrointestinal simulation.

Memory impairment in rats by hippocampal administration of the serine protease subtilisin.

Since the serine protease subtilisin has been reported to generate a novel form of long-term depression (LTD) in rat hippocampal slices, the present work was designed to determine whether it has any effect on learning and memory processes. Rats were used to examine the effects of subtilisin, injected directly into the dorsal hippocampus, on task performance in a step-through inhibitory avoidance of a mild footshock. The administration of 100 ng of subtilisin into each hippocampus, immediately after training, was sufficient to induce a detectable learning deficit with a footshock stimulus of 0.5 mA. Higher doses produced dose-related impairments in memory consolidation. These effects were not the result of irreversible toxicity, since rats trained with a higher amplitude footshock (0.75 mA) were able to perform as control animals; therefore, the amnesic effect was not further evident. Furthermore, the administration of subtilisin before avoidance training did not produce any detectable effect on performance during the training or test sessions, indicating that neither acquisition nor consolidation was affected. It is concluded that the post-training administration of a serine protease inhibitor is able to produce robust deficits of memory consolidation consistent with its ability to generate LTD, raising the possibility that related molecules could play physiological or pathological roles in the modulation of learning and memory.

Effect of sodium hypochlorite with the addition of a proteolytic enzyme on postoperative discomfort: a multicenter randomized clinical trial.

AIM: The purpose of this study was to compare the patient's postoperative discomfort when root canal irrigation was performed either with standard sodium hypochlorite or with sodium hypochlorite with the adjunct of a proteolytic enzyme. METHODS: Two hundred patients were endodontically treated in two clinics. The type of irrigant to be used during root canal instrumentation was randomly assigned. Final irrigation was done using EDTA 17%. The canals were filled by warm vertical condensation with guttha-percha and the coronal seal was made using IRM. Patients were given a questionnaire to assess pain and swelling and the number of analgesics and other drugs taken during the first week after treatment. RESULTS: A total of 166 questionnaires could have been evaluated. No significant difference was found between groups for pain, swelling and analgesics taken. Moderate pain and swelling was reported only in the first two days after treatment. No antibiotics use was reported. No guttha-percha excess beyond root apex was found by radiographic assessment. CONCLUSIONS: The irrigating solution containing a proteolytic enzyme does not produce greater postoperative discomfort as compared to the conventional sodium hypochlorite in patients undergoing endodontic therapy.

Encapsulation of crosslinked subtilisin microcrystals in hydrogel beads for controlled release applications.

Enzymes are less stable in harsh conditions and hence to overcome this nature, several methodologies are being developed. It was found that crosslinked enzyme crystals are the most promising strategy for the stabilization of the enzymes [Emilia Abraham, T., Jegan Roy, J., Bindhu, L.V., Jayakumar, K.K., 2004. Crosslinked enzyme crystals of glucoamylase as a potent catalyst for biotransformations. Carbohydr. Res. 339, 1099-1104; Navia, M., St. Clair, N., 1997. Crosslinked enzyme crystals. Biosens. Bioelectron. 12, 7]. A cost effective methodology of crystallization of protease (Bacillus subtilis) with ammonium sulphate (65%, w/v) and then crosslinking the crystals with glutaraldehyde (4%, v/v) in isopropanol for 20min gave a stable and active enzyme. SEM studies showed that the protease is in small cubic shaped crystals of 1-2 microm size. Crosslinked enzyme crystal (CLEC) of protease has good stability in polar and nonpolar organic solvents, such as hexane, toluene, benzene and carbon tetrachloride and it had high thermal stability up to 60 degrees C and hence can be used as a catalyst for the biotransformation of compounds which are not soluble in aqueous medium. The CLECs were entrapped in the alginate:guar gum (3:1) composite beads which were resistant to low pH conditions in the stomach and hence was found to be useful for the oral drug delivery. This method can be used to deliver the protein and peptide drugs which require high concentrations at the delivery stage, and which normally degrades in the stomach before reaching the jejunum. Application of these pH-sensitive beads for the controlled release of subtilisin in in vitro was studied and found to be a viable strategy.

A silicone-based controlled-release device for accelerated proteolytic debridement of wounds.

A new device for rapid enzymatic debridement of cutaneous wounds has been developed using a controlled-release, silicone-based, dried emulsion. A dehydrated serine protease of the subtilisin family, previously untested for wound debridement, was incorporated into the emulsion. This device exhibited excellent storage stability. Moisture from the wound triggered an even, reproducible, and complete release of the enzyme within the first 8 hours. The device maintains a moist wound environment that allows the enzyme to achieve nearly complete digestion of the hardened eschar of full-thickness burns in a porcine model after an exposure period of 24 hours. Debridement was faster than in untreated wounds or wounds treated with a currently available enzyme ointment. Following rapid enzymatic debridement, healing appeared to progress normally, with no histological evidence of damage to adjacent healthy tissue.

Topical protease therapy as a novel method of epidermal ablation: preliminary report.

BACKGROUND: For more than 50 years, proteolytic enzymes have been extensively used in laboratory settings for the purposes of in vitro epidermal separation and keratinocyte isolation. However, the topical, in vivo pharmacologic properties of these enzymes are virtually unknown. Previous therapeutic applications for topically applied proteases have been limited to wound debridement. OBJECTIVE: To characterize the clinical and histologic effects of topically applied proteases as a method of therapeutic epidermal ablation. MATERIALS AND METHODS: SKH-1 hairless mouse and human skin samples were exposed both in vitro and in vivo to varying concentrations of the proteases subtilisin, trypsin, and dispase for different exposure durations. The effects of protease exposure were then assessed by both clinical and histologic examination. RESULTS: Exposure of both human and mouse skin samples to topical protease solutions resulted in reproducible, differential patterns of epidermal ablation: subcorneal, intraepidermal, and subepidermal. In a limited study, topical application of trypsin solution resulted in the scar-free ablation of lesions of seborrheic keratosis located on the lower extremity. CONCLUSION: Topically applied proteases represent an alternative method of epidermal ablation with several potential advantages over existing techniques. Further studies are needed to delineate ideal enzyme formulations, vehicles, and applications.