Lumbrokinase/paclitaxel nanoparticle complex: potential therapeutic applications in bladder cancer.

BACKGROUND: Lumbrokinase (LK) is an enzyme complex with antithrombotic, antioxidant, antitumor, and immunomodulatory effects. It has been extensively studied and used in clinical anti-tumor therapy. However, its half-life is short, its bioavailability is low, and its toxicity and side effects are great, which greatly limit its clinical application. Therefore, LK is often combined with other drugs (such as immune agents, hormones, or Chinese herbal medicine) to reduce its dosage and side effects and to improve its anti-tumor effects. METHODS AND RESULTS: Here, we described an LK/paclitaxel (PTX) nanocarrier based on poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)) (PEG-b-(PELG-g-(PZLL-r-PLL))). In the present study, LK and PTX were loaded by electrostatic and/or hydrophobic effects under mild conditions, thereby increasing the half-life and bioavailability of the drugs via the sustained release and enhancement of tumor site enrichment by the LK/PTX/PEG-b-(PELG-g-(PZLL-r-PLL)) complex through passive targeting. In this study, using bladder cancer cells (J82 cells) and rat bladder cancer model as the object, the structure of the nanocarrier, the relationship between drugs composition and antitumor properties were systematically studied. CONCLUSION: We propose that the block copolymer PEG-b-(PELG-g-(PZLL-r-PLL)) may function as a potent nanocarrier for augmenting anti-bladder cancer pharmacotherapy, with unprecedented clinical benefits.

Effects of Phage Endolysin SAL200 Combined with Antibiotics on Staphylococcus aureus Infection.

Phages and their derivatives are increasingly being reconsidered for use in the treatment of bacterial infections due to the rising rates of antibiotic resistance. We assessed the antistaphylococcal effect of the endolysin SAL200 in combination with standard-of-care (SOC) antibiotics. The activity of SAL200 when it was combined with SOC antibiotics was assessed in vitro by checkerboard and time-kill assays and in vivo with murine bacteremia and Galleria mellonella infection models. SAL200 reduced the SOC antibiotic MICs and showed a ≥3-log10-CFU/ml reduction of Staphylococcus aureus counts within 30 min in time-kill assays. Combinations of SAL200 and SOC antibiotics achieved a sustained decrease of >2 log10 CFU/ml. SAL200 significantly lowered the blood bacterial density within 1 h by >1 log10 CFU/ml in bacteremic mice (P < 0.05 versus untreated mice), and SAL200 and SOC antibiotic combinations achieved the lowest levels of bacteremia. The bacterial density in splenic tissue at 72 h postinfection was the lowest in mice treated with SAL200 and SOC antibiotic combinations. SAL200 combined with SOC antibiotics also improved Galleria mellonella larva survival at 96 h postinfection. The combination of the phage endolysin SAL200 with SOC antistaphylococcal antibiotics showed synergistic effects in vitro and in vivo The combination of SAL200 with SOC antibiotics could help in the treatment of difficult-to-treat S. aureus infections.

Development of Phage Lysins as Novel Therapeutics: A Historical Perspective.

Bacteriophage lysins and related bacteriolytic enzymes are now considered among the top antibiotic alternatives for solving the mounting resistance problem. Over the past 17 years, lysins have been widely developed against Gram-positive and recently Gram-negative pathogens, and successfully tested in a variety of animal models to demonstrate their efficacy. A lysin (CF-301) directed to methicillin resistant Staphylococcus aureus (MRSA) has effectively completed phase 1 human clinical trials, showing safety in this novel therapeutic class. To validate efficacy, CF-301 is currently the first lysin to enter phase 2 human trials to treat hospitalized patients with MRSA bacteremia or endocarditis. If successful, it could be the defining moment leading to the acceptance of lysins as an alternative to small molecule antibiotics. This article is a detailed account of events leading to the first therapeutic use and ultimate development of phage-encoded lysins as novel anti-infectives.

Are Phage Lytic Proteins the Secret Weapon To Kill Staphylococcus aureus?

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most threatening microorganisms for global human health. The current strategies to reduce the impact of S. aureus include a restrictive control of worldwide antibiotic use, prophylactic measures to hinder contamination, and the search for novel antimicrobials to treat human and animal infections caused by this bacterium. The last strategy is currently the focus of considerable research. In this regard, phage lytic proteins (endolysins and virion-associated peptidoglycan hydrolases [VAPGHs]) have been proposed as suitable candidates. Indeed, these proteins display narrow-spectrum antimicrobial activity and a virtual lack of bacterial-resistance development. Additionally, the therapeutic use of phage lytic proteins in S. aureus animal infection models is yielding promising results, showing good efficacy without apparent side effects. Nonetheless, human clinical trials are still in progress, and data are not available yet. This minireview also analyzes the main obstacles for introducing phage lytic proteins as human therapeutics against S. aureus infections. Besides the common technological problems derived from large-scale production of therapeutic proteins, a major setback is the lack of a proper legal framework regulating their use. In that sense, the relevant health authorities should urgently have a timely discussion about these new antimicrobials. On the other hand, the research community should provide data to dispel any doubts regarding their efficacy and safety. Overall, the appropriate scientific data and regulatory framework will encourage pharmaceutical companies to invest in these promising antimicrobials.

Measuring Direct Oral Anticoagulants.

Direct oral anticoagulants (DOACs) can be quantified using methods that can be performed in any clinical or research laboratory using manual or automated instrument platforms. Dabigatran etexilate, the oral direct thrombin inhibitor, can be quantified by drug-calibrated clot or chromogenic-based assays using either thrombin or ecarin as substrates. Oral direct anti-Xa inhibitors, such as rivaroxaban, apixaban, and edoxaban, can be quantified with drug-calibrated anti-Xa kits or reagents as typically used for measuring heparins (unfractionated, low molecular weight, or pentasaccharides).

Antioxidant and antithrombotic therapies for diabetic kidney disease.

With an increasing incidence, diabetic kidney disease (DKD) has been the leading cause of chronic kidney disease and end-stage renal disease, and conventional therapies did not change this situation. This study intended to review and analyze the antioxidant and antithrombotic treatments of DKD for seeking novel therapeutic strategies. Relevant articles involved with antioxidant and antithrombotic treatments in DKD were retrieved and analyzed via systematic assessment. Meta-analysis showed that pancreatic kallikrein definitely reduced glycated hemoglobin in DKD patients (mean difference, 0.36%; 95% confidence interval, 0.08% to 0.63%; P = .01). Apart from the classic agents such as aspirin, novel drugs such as pancreatic kallikrein, sulodexide, and especially the traditional Chinese medicine including Tripterygium wilfordii and lumbrukinase, exert beneficial effects in DKD patients. Antioxidant and antithrombotic treatments are beneficial for DKD patients and represent promising therapeutic strategies in the future.

Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection.

OBJECTIVES: In the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins [peptidoglycan hydrolases (PGHs)] have been suggested as promising antimicrobial agents. The aim of this study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs. METHODS: PGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park-Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model. RESULTS: Despite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by evolutionarily divergent catalytic domains), the enzymes displayed varying degrees of in vitro lytic activity against numerous staphylococcal strains, including cell surface mutants and drug-resistant strains, and proved effective against static biofilms. In a mouse model of systemic MRSA infection, six PGHs provided 100% protection from death, with animals being free of clinical signs at the end of the experiment. CONCLUSIONS: Our results corroborate the high potential of PGHs for treatment of S. aureus infections and reveal unique antimicrobial and biochemical properties of the different enzymes, suggesting a high diversity of potential applications despite highly conserved peptidoglycan target sites.

Thrombolytic effects in vivo of nattokinase in a carrageenan-induced rat model of thrombosis.

BACKGROUND/AIMS: Nattokinase is a serine protease produced by Bacillus subtilis during the fermentation of the soybean product natto. The fibrinolytic activity and thrombolytic effects of nattokinase have been observed in vitro, but the effect in vivo has still to be researched. The objective of this study was to demonstrate the activity of nattokinase in vivo. METHODS: To establish a rat model of thrombosis, κ-carrageenan was injected subcutaneously into the toes of Sprague-Dawley (SD) rats. Histological examination confirmed thrombosis. The rats were then treated with varying doses of nattokinase and the resulting thrombolysis was histologically assessed. ELISA was used to determine the levels of the fibrin/fibrinogen degradation products (FDPs) and D-dimer, which are sensitive indices of fibrinolytic activity. Vermis kinase, a known thrombolytic agent, was used as a positive control. RESULTS: Biopsy results revealed partial thrombolysis in the tail vessels of the rats treated with nattokinase or vermis kinase. FDP and D-dimer levels were higher in rats treated with high-dose nattokinase than in those treated with saline. No difference in FDP or D-dimer levels was observed between rats treated with high-dose nattokinase and those treated with vermis kinase. CONCLUSIONS: Both the histological and physiological evidence from this study indicate that nattokinase exerts thrombolytic effects in vivo.

Antibacterial bioagents based on principles of bacteriophage biology: an overview.

Bacteriophages were discovered almost a century ago. With the advent of antibiotics, the use of bacteriophages for treatment of infections fell out of favor in Western medicine. In light of the rise of antibiotic resistance, phages and their products (lysins) are rediscovered as antibacterial bioagents. This overview summarizes principles of phage biology and their translation for therapeutic and preventive applications. Examples are presented to highlight their therapeutic promise for prophylaxis and treatment of bacterial infections including multidrug-resistant organisms in humans and animals, and their use as decontaminants of food supplies and environments. Besides research on the in vivo behavior of phages and lysins, dialogues between researchers and regulatory agencies are necessary to publish guidelines for bacteriophage manufacturing and formulation for human use. Only well-designed, double-blind randomized controlled trials will determine if phages and lysins are safe and effective adjuncts or alternatives to antibiotic therapy for infections with multidrug-resistant organisms.

A prospective, randomized, double-blind, placebo-controlled clinical trial comparing the efficacy of systemic enzyme therapy for edema control in orthognathic surgery using ultrasound scan to measure facial swelling.

PURPOSE: To evaluate the effectiveness of systemic enzyme therapy for the control of edema in patients who undergo bimaxillary orthognathic surgery. MATERIALS AND METHODS: Thirty patients were included in this double-blinded, randomized, control trial. Before surgery, each patient was allotted a code (study or control group). Nine anthropometric points were selected. Thickness of the soft tissue at each of these points was measured using an ultrasound device. These measurements were performed on the day before surgery and 1, 5, and 15 days after surgery. The study group was given a twice-daily dose of systemic enzyme therapy from the first postoperative day for 5 days; the control group was given placebo. The percentage of difference in the thickness of the soft tissue was calculated at each of the 9 points on postoperative days 1, 5, and 15. These data were analyzed and compared using the Mann-Whitney test. RESULTS: The statistical evaluation showed a significant difference in soft tissue thickness between the 2 groups, especially on days 5 and 15, at most assessed points. CONCLUSION: The results of this study suggest that systemic enzyme therapy significantly decreases postoperative edema in orthognathic surgery, precluding long-term corticosteroid use.

Targeted modification of wheat grain protein to reduce the content of celiac causing epitopes.

The prolamin peptides in wheat gluten and in the homologous storage proteins of barley and rye cause painful chronic erasure of microvilli of the small intestine epithelium in celiac patients. If untreated, it can lead to chronic diarrhea, abdominal distension, osteoporosis, weight-loss due to malabsorption of nutrients, and anemia. In addition to congenital cases, life-long exposure to gluten proteins in bread and pasta can also induce development of celiac sprue in adults. To date, the only effective treatment is life-long strict abstinence from the staple food grains. Complete exclusion of dietary gluten is, however, difficult due to use of wheat in many foods, incomplete labeling and social constraints. Thus, finding alternative therapies for this most common foodborne disease remained an active area of research, which has led to many suggestions in last few years. The pros and cons associated with these therapies were reviewed in the present communication. As different celiac patients are immunogenic to different members of the undigestible proline/glutamine rich peptides of ~149 gliadins and low molecular weight glutenin subunits as well as the six high molecular weight glutenin subunits, an exhaustive digestion of the immunogenic peptides in the stomach, duodenum, jejunum, and ileum of celiacs is required. In view of the above, we evaluated the capacity of cereal grains to synthesize and store the enzymes prolyl endopeptidase from Flavobacterium meningosepticum and the barley cysteine endoprotease B2, which in combination are capable of detoxifying immunogenic gluten peptides in a novel treatment of celiac disease.

Potential of bacteriophages and their lysins in the treatment of MRSA: current status and future perspectives.

Bacteriophages (phages) are viruses that specifically infect and kill bacteria. Lysins are enzymes of bacteriophage origin that cleave covalent bonds in peptidoglycan, thereby inducing rapid lysis of a bacterial cell. As potential antibacterial agents, phages and lysins have some important features in common, especially the capacity to kill antibiotic-resistant bacteria, a narrow antibacterial range, and lack of toxic effects on mammalian cells. In this article we present the staphylococcal phages and their lysins that can be used to combat methicillin-resistant Staphylococcus aureus (MRSA), one of today's most dangerous pathogens. We also discuss the use of phages as vectors specifically delivering different antibacterial agents to bacterial cells. Experimental data show that both phages and lysins could be effective in the treatment of MRSA.

Improved myocardial perfusion in stable angina pectoris by oral lumbrokinase: a pilot study.

OBJECTIVE: A novel antithrombotic agent, lumbrokinase, was evaluated in this pilot study for its efficacy in the treatment of symptomatic stable angina. DESIGN: This was a single-armed cohort study. SETTINGS: The study was conducted at the National Cardiovascular Center, Harapan Kita, Jakarta, Indonesia. SUBJECTS: The study comprised 10 patients who had coronary artery disease and stable angina and who consented to the trial. INTERVENTION: Patients were treated with oral lumbrokinase for 30 consecutive days in addition to their standard medical therapy. OUTCOME MEASURES: Stress technetium-99m sestamibi myocardial perfusion imaging (MPI) was performed before and at the conclusion of the active treatment period. The degree and extent of inducible ischemia observed on the myocardial perfusion images were evaluated using the previously validated semiquantitative indices (Summed Stress Score and Summed Difference Score). RESULTS: Following active treatment, the mean Summed Stress Score and Summed Difference Score deceased by 39% and 37%, respectively. The anginal symptom was ameliorated in 6 of 10 patients. No adverse reaction including major or minor bleeding was observed. CONCLUSIONS: This paper represents the first description of the use of oral lumbrokinase in the treatment of chronic coronary artery disease with objective assessment using MPI. Oral lumbrokinase improves regional myocardial perfusion in patients with stable angina.

[Protective effects of Chailing Guiqi Decoction combined with lumbrukinase on renal function in rats with adriamycin nephropathy].

OBJECTIVE: To study the protective efffects of Chailing Guiqi Decoction (CLGQD) combined with lumbrukinase on renal function in rats with adriamycin nephropathy. METHODS: Thirty-six SD rats were randomly divided into four groups: normal control group, untreated group, simvastatin-treated group and CLGQD -treated group. Adriamycin nephropathy was induced by intravenous injection with 5 mg/kg adriamycin. After seven-day treatment, quantitative measurement of 24-h urine protein was determined with trichloroacetic acid, and serum total protein (TP), albumin (Alb), total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), creatinine (Cr) and blood urea nitrogen (BUN) were assessed using automatic biochemistry analyzer. The pathomorphological changes of renal tissues were observed with light and electron microscopes. RESULTS: In the untreated group, the 24-h urine protein excretion, serum TC, TG, LDL, Cr and BUN were significantly higher than those in the normal control group (P<0.05 or P<0.01), while the serum TP, Alb, HDL were significantly lower than those in the normal control group (P<0.01). In the CLGQD-treated group, the 24-h urine protein excretion, serum TC, TG, LDL, Cr and BUN were significantly lower as compared with those in the untreated group (P<0.05 or P<0.01), while the serum TP, Alb and HDL were significantly higher as compared with those in the untreated group (P<0.05 or P<0.01). The pathomorphological findings of the renal tissues under the light microscope in the untreated group showed focal segmental glomerulosclerosis in a few of glomerulus, degenerated and swelled proximal tubular epithelial cells, proteins in cast formation in some renal tubules and scattered fibrosis in interstitial tissues of the kidney, while the electron microscope images showed the fusion of foot processes in glomerular epithelial cells. The pathomorphological changes in the CLGQD-treated group were slighter than those in the untreated group. CONCLUSION: CLGQD combined with lumbrukinase can reduce proteinuria, regulate lipid metabolism, protect renal function, and delay progressive renal damage in rats.

Impact of complementary oral enzyme application on the postoperative treatment results of breast cancer patients--results of an epidemiological multicentre retrolective cohort study.

PURPOSE: [corrected] To evaluate the impact of postoperative treatment with an oral enzyme (OE) preparation given complementary to an antineoplastic therapy in patients with breast cancer. METHODS: The design of this epidemiological study was a retrolective cohort analysis with parallel groups. Design and conduct of the study were performed to current standards for prospective, controlled clinical trials. A cohort of 2,339 breast cancer patients undergoing surgical intervention and radio-, chemo- or hormonal therapy were studied in 216 centres. Of the 2,339 patients, 1,283 received complementary treatment with OE and 1,056 did not receive OE. Patients with other complementary medications were excluded and the final analysis was performed with the data from 649 patients, of whom 239 (37%) were additionally treated with OE (test group) and 410 (63%) without OE (control group). The median follow-up time for the test group was 485 days and for the control group 213 days. The primary endpoint of the study was to determine whether complementary treatment with OE can reduce typical disease- or therapy-associated signs and symptoms (gastrointestinal symptoms, mental symptoms, dyspnoea, headache, tumour pain, cachexia, skin disorders, infections, and side effects associated with the antineoplastic therapy) in patients with breast cancer. Imbalances for causal effects (covariates) were adjusted for by means of the propensity score. Outcome analysis was performed by estimating the linear regression between change in symptom score and propensity score with all data and using this regression line to calculate the change in symptom score which would be expected for each patient. Tumour-associated events (recurrence, metastasis, and death) were evaluated in terms of the number of events observed and time to event. The safety of treatment with OE was analysed in terms of the number and severity of adverse events, their duration, treatment and outcome. RESULTS: For all symptoms except tumour pain, the adjusted mean improvement in symptom scores was larger in the test group than in the control group. The adjusted difference was statistically significant for all symptoms, except tumour pain and infections. The results show that the typical disease- and therapy-associated signs and symptoms in patients on complementary therapy with OE during postoperative treatment were significantly less. For 75% of the test group and 55% of the control group the physician recorded "no signs and symptoms". A clear reduction in the side effects of radiotherapy and chemotherapy was documented in 74% of the test group and 55% of the control group. Analysis of survival, recurrence, and metastasis demonstrated a reduced number of events in the test group. There was evidence of a beneficial influence of OE on time to event, although the median observation time was too short in these breast cancer patients to draw definite conclusions. The safety component was judged in 98% of the test group and 76% of the control group as "very good" or "good". In the total sample of 2,339 patients, the rate of OE-associated adverse reactions was 3.2%. All side effects were mild to moderate gastrointestinal symptoms. CONCLUSION: Complementary treatment of breast cancer patients with OE improves the quality of life by reducing signs and symptoms of the disease and the side effects of adjuvant antineoplastic therapies. This epidemiological retrolective cohort analysis provides evidence that the patients may also gain benefit by a prolongation of the time to event for cancer recurrence, metastasis and survival. OE was generally well tolerated.

Novel clinical strategies for the treatment of pancreatic carcinoma.

Pancreatic carcinoma ranks as the eighth most frequent type of solid tumour arising worldwide yet it represents the fourth most frequent cause of death. This discrepancy reflects the current lack of effective treatment available for the pancreatic cancer patient and highlights the urgent need for new therapeutic principles in this area. The last five years have seen an increasing number of novel approaches both in the pre-clinical area as well as in clinical trials for pancreatic cancer treatments. This review summarizes these new developments and attempts to rationalize the possibilities available for the patient at the beginning of the new millennium.

Therapy with proteolytic enzymes in rheumatic disorders.

Plant extracts with a high content of proteolytic enzymes have been used in traditional medicine for a long time. Besides herbal proteinases, 'modern' enzyme therapy includes pancreatic enzymes. The therapeutic use of proteolytic enzymes is empirically based, but is also supported by scientific studies. This review provides an overview of preclinical and clinical trials of systemic enzyme therapy in rheumatic disorders. Studies of the use of proteolytic enzymes in rheumatic disorders have mostly been carried out on enzyme preparations consisting of combinations of bromelain, papain, trypsin and chymotrypsin. The precise mechanism of action of systemic enzyme therapy remains unresolved. The ratio of proteinases to antiproteinases, which is affected by rheumatic diseases, appears to be influenced by the oral administration of proteolytic enzymes, probably via induction of the synthesis of antiproteinases or a signal transduction of the proteinase-antiproteinase complex via specific receptors. Furthermore, there are numerous alterations of cytokine composition during therapy with orally administered enzymes resulting from immunomodulatory effects, which might be an indication of the efficacy of enzyme therapy. The results of various studies (placebo-controlled and comparisons with nonsteroidal anti-inflammatory drugs) in patients with rheumatic diseases suggest that oral therapy with proteolytic enzymes produces certain analgesic and anti-inflammatory effects. However, the results are often inconsistent. Nevertheless, in the light of preclinical and experimental data as well as therapeutic experience, the application of enzyme therapy seems plausible in carefully chosen patients with rheumatic disorders.