Platelet activation and endothelial dysfunction biomarkers in acute coronary syndrome: the impact of PCSK9 inhibition.

AIMS: Platelet activation and endothelial dysfunction contribute to adverse outcomes in patients with acute coronary syndromes (ACS). The goals of this study were to assess the impact of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition on markers of platelet activation and endothelial dysfunction in ACS patients and the interaction among PCSK9, platelets, and endothelial cells (ECs) on left internal mammary artery (LIMA) vascular endothelium using specimens obtained during coronary artery bypass surgery (CABG). METHODS AND RESULTS: Acute coronary syndromes patients enrolled in the Evolocumab in ACS trials were randomized to placebo or a single dose of 420 mg evolocumab within 24 h of hospitalization. Serum samples for analysis of platelet factor 4 (PF4) and P-selectin, markers of platelet activation, and von Willebrand factor (vWF), a marker of endothelial dysfunction, were obtained at baseline and 30 days. Additionally, LIMA segments obtained during CABG from patients who were and were not receiving evolocumab were immunostained with PCSK9; CD61, a platelet-specific marker; and CD31, an endothelial cell-specific marker. Forty-six participants were randomized to placebo or to evolocumab. Controlling for baseline levels, PF4 and vWF were significantly lower in the evolocumab, than in the placebo, group at 30 days. Immunostaining of LIMA specimens from twelve participants undergoing CABG revealed colocalization of PCSK9, CD61, and CD31 at the vascular endothelium. Administration of evolocumab was associated with decreased overlap of PCSK9, CD61, and CD31. CONCLUSIONS: Proprotein Convertase Subtilisin/Kexin 9 inhibition decreases markers of platelet activation and endothelial dysfunction in ACS patients. PCSK9 is associated with platelets and vascular ECs in LIMA segments and PCSK9 inhibition decreases that interaction.

Nattokinase prevents ß-amyloid peptide (Aß1-42) induced neuropsychiatric complications, neuroinflammation and BDNF signalling disruption in mice.

Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disorder characterized by abnormal accumulation of extracellular ß-amyloid (Aß) plaques and neuronal damage. Although AD is typically considered a cognitive neurodegenerative disorder, almost all people diagnosed with AD develop neuropsychiatric complications at some stage in their life span. The present study investigated the effect of chronic Nattokinase (NK) administration on ß-Amyloid peptide (Aß1-42) induced neuropsychiatric conditions (depression-like behaviour, anxiety, and memory impairment) in mice. Aß1-42 peptide injected mice demonstrated depression, anxiety, and impairment of cognitive abilities evaluated as increased immobility time in forced swim test (FST), decreased open arm time/entries in elevated plus maze (EPM) and reference and working memory error in radial arm maze (RAM) respectively with elevation in Interleukin-6 (IL-6), Tumour necrosis factor-α (TNF-α), reduction in Interleukin-10 (IL-10) and Brain-derived neurotrophic factor (BDNF) immunocontent within the hippocampus. Chronic administration of NK (50-100 mg/kg, i.p.) from day 8-27, prevented depression-like behaviour, anxiety, and memory impairment and normalized the neurochemical alteration within the hippocampus of mice injected with Aß1-42 peptide. The effect of NK on psychiatric complications, learning, and memory was comparable to peripheral donepezil treatment. This study suggests that NK improves learning, memory impairment, and neuropsychiatric complications possibly through the downregulation of neuroinflammatory pathways and restoring BDNF signalling in AD.

Safety and Effectiveness of Alirocumab, a Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitor, in Patients With Familial or Non-Familial Hypercholesterolemia　- A Post-Marketing Survey (J-POSSIBLE).

BACKGROUND: This study evaluated the safety and effectiveness of alirocumab in Japanese patients with familial hypercholesterolemia (FH) or non-FH in a real-world clinical setting.Methods and Results: This post-marketing surveillance study had a 2-year standard observation period. The study included Japanese patients with hypercholesterolemia who were treatment naïve to alirocumab, had a high risk of developing cardiovascular events, and had an insufficient response to, or were unsuitable for, treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Alirocumab was administered at a dose of 75 or 150 mg via subcutaneous injection every 2 or 4 weeks. Overall, 1,177 and 1,038 patients were included in the safety and effectiveness analysis populations, respectively. The incidence of adverse drug reactions (ADRs) was 3.4% (40/1,177). The time to ADR occurrence was within 4 weeks in half the patients experiencing ADRs (n=20). There were no meaningful differences in the ADRs experienced in the FH and non-FH groups. The mean (±SE) percentage changes in low-density lipoprotein cholesterol from baseline to last observation carried forward were -46.9±2.1% and -42.7±2.0% in the non-FH and FH groups, respectively. Total cholesterol, triglycerides, apolipoprotein B/E, and lipoprotein(a) concentrations were decreased at Week 4 and maintained until Week 104 in the overall population. CONCLUSIONS: Alirocumab was well tolerated and showed effectiveness in Japanese patients with hypercholesterolemia in a real-world clinical setting.

[Comparison of the efficacy of PCSK9 inhibitors and statins on dyslipidemia in patients with type 2 diabetes].

Objective: To compare the efficacy of preprotein convertase subtilisin lysozyme 9 (PCSK9) inhibitors with statins in patients with type 2 diabetes mellitus (T2DM). Methods: A total of 140 patients with T2DM (80 males and 60 females) in the People's Hospital Affiliated to Shandong First Medical University from January 2018 to January 2021 were selected, with a mean age of (55±5) years (41-72 years). The patients were divided into observation group (n=68) and control group (n=72) by the random number table method. Both groups were given conventional treatments such as hypoglycemic drugs, the control group was given statins to regulate lipids, and the observation group was given PCSK9 inhibitors to lower lipids. The differences of low-density lipoprotein cholesterol (LDL-C), interleukin (IL)-1, IL-6, IL-8, IL-10, tumor necrosis factor-α (TNF-α), C-reactive protein (CRP) expression levels and standard-reaching rate of LDL-C between the two groups were compared. The correlation between serum PCSK9 level and fasting plasma glucose (FPG), hemoglobin A1c (HbA1c) and other indicators in T2DM patients was analyzed by Pearson correlation analysis. Results: After treatment, the LDL-C of the observation group was (2.3±0.7) mmol/L, which was lower than that of the control group [(2.7±0.7) mmol/L] (P=0.024); the standard-reaching rate of LDL-C of the observation group was 89.7% (61/68), which was higher than that of the control group [68.1% (49/72)] (P=0.002); the levels of IL-1, IL-6, TNF-, CRP, IL-10 and IL-8 in the observation group after treatment were (27.6±6.6) ng/L, (36.7±6.9) ng/L, (40.1±8.9) ng/L, (7.8±1.8) ng/L, (19.2±3.3) ng/L, (13.7±3.3) ng/L, respectively, which were lower than those in the control group [(30.6±7.9) ng/L, (40.1±7.3) ng/L, (43.4±9.2) ng/L, (10.4±2.5) ng/L, (30.7±3.7) ng/L, (26.8±3.4) ng/L, respectively] (all P<0.05). After treatment, the PCSK9 level in the observation group was (74±13) µg/L, which was lower than that in the control group [(97±14) µg/L] (P<0.001). The level of PCSK9 in T2DM patients was positively correlated with LDL-C, IL-1, IL-6 and TNF-α (r=0.390, 0.433, 0.398 and 0.562, all P<0.05). Conclusion: PCSK9 inhibitors have better lipid-regulating effects in patients with T2DM and can improve the level of inflammation at the same time.

Safety and efficacy of inclisiran in South African patients at high cardiovascular risk: A subanalysis of the ORION phase III clinical trials.

BACKGROUND: Inclisiran significantly reduced low-density lipoprotein cholesterol (LDL-C) in individuals with heterozygous familial hypercholesterolaemia, established atherosclerotic cardiovascular disease (ASCVD) or ASCVD risk equivalents (type 2 diabetes, familial hypercholesterolaemia or a 10-year risk of a cardiovascular event ≥20%) in the ORION phase III clinical trials. Infrequent dosing at days 1, 90, 270 and 450 resulted in a mean LDL-C reduction of ~50%. A total of 298 participants from South Africa (SA) were enrolled. Local data are needed to support the use of inclisiran in the SA population, potentially addressing an unmet need for additional LDL-C-lowering therapies. Objectives. To analyse the ORION phase III trial data to assess the efficacy and safety of inclisiran in SA participants. Methods. ORION-9, 10 and 11 were randomised, double-blind, phase III trials. Participants were receiving maximally tolerated statins with or without other lipid-lowering therapies (excluding protein convertase subtilisin/kexin type 9 (PCSK9) inhibitors). Participants were randomised 1:1 to inclisiran sodium 300 mg/284 mg (free acid) or placebo administered at days 1, 90, 270 and 450. The co-primary endpoints were the LDL-C percentage change from baseline to day 510 and the time-averaged percentage change in LDL-C from baseline after day 90 up to day 540. Key secondary endpoints included the absolute change in LDL-C from baseline to day 510, the time-averaged absolute change from baseline after day 90 up to day 540, and changes in other lipids and lipoproteins. Results. The mean age of the participants was 58.6 years (56% male). The mean LDL-C level at baseline was 3.6 mmol/L. At day 510, inclisiran reduced LDL-C levels by 54.2% compared with placebo (95% confidence interval (CI) -61.3 - -47.2; p<0.0001). The corresponding time-averaged reduction in LDL-C was 52.8% (95% CI -57.9 - -47.8; p<0.0001). Treatment-emergent adverse events at the injection site were more common with inclisiran compared with placebo (10.1% v. 0.7%); however, all were mild or moderate in nature and none were persistent. Conclusion. Inclisiran, given in addition to maximally tolerated standard lipid-lowering therapy, is effective and safe and results in robust reductions in LDL-C in SA patients at high cardiovascular risk.

Colivelin, a synthetic derivative of humanin, ameliorates endothelial injury and glycocalyx shedding after sepsis in mice.

Endothelial dysfunction plays a central role in the pathogenesis of sepsis-mediated multiple organ failure. Several clinical and experimental studies have suggested that the glycocalyx is an early target of endothelial injury during an infection. Colivelin, a synthetic derivative of the mitochondrial peptide humanin, has displayed cytoprotective effects in oxidative conditions. In the current study, we aimed to determine the potential therapeutic effects of colivelin in endothelial dysfunction and outcomes of sepsis in vivo. Male C57BL/6 mice were subjected to a clinically relevant model of polymicrobial sepsis by cecal ligation and puncture (CLP) and were treated with vehicle or colivelin (100-200 µg/kg) intraperitoneally at 1 h after CLP. We observed that vehicle-treated mice had early elevation of plasma levels of the adhesion molecules ICAM-1 and P-selectin, the angiogenetic factor endoglin and the glycocalyx syndecan-1 at 6 h after CLP when compared to control mice, while levels of angiopoietin-2, a mediator of microvascular disintegration, and the proprotein convertase subtilisin/kexin type 9, an enzyme implicated in clearance of endotoxins, raised at 18 h after CLP. The early elevation of these endothelial and glycocalyx damage biomarkers coincided with lung histological injury and neutrophil inflammation in lung, liver, and kidneys. At transmission electron microscopy analysis, thoracic aortas of septic mice showed increased glycocalyx breakdown and shedding, and damaged mitochondria in endothelial and smooth muscle cells. Treatment with colivelin ameliorated lung architecture, reduced organ neutrophil infiltration, and attenuated plasma levels of syndecan-1, tumor necrosis factor-α, macrophage inflammatory protein-1α and interleukin-10. These therapeutic effects of colivelin were associated with amelioration of glycocalyx density and mitochondrial structure in the aorta. At molecular analysis, colivelin treatment was associated with inhibition of the signal transducer and activator of transcription 3 and activation of the AMP-activated protein kinase in the aorta and lung. In long-term outcomes studies up to 7 days, co-treatment of colivelin with antimicrobial agents significantly reduced the disease severity score when compared to treatment with antibiotics alone. In conclusion, our data support that damage of the glycocalyx is an early pathogenetic event during sepsis and that colivelin may have therapeutic potential for the treatment of sepsis-associated endothelial dysfunction.

Paediatric patients with heterozygous familial hypercholesterolaemia treated with evolocumab for 80 weeks (HAUSER-OLE): a single-arm, multicentre, open-label extension of HAUSER-RCT.

BACKGROUND: The HAUSER-RCT study showed that 24 weeks of evolocumab (a proprotein convertase subtilisin/kexin type 9 [PCSK9] inhibitor) in paediatric patients with heterozygous familial hypercholesterolaemia was safe and improved lipid parameters compared to placebo. Here, we aimed to evaluate the safety and efficacy of evolocumab in this population for an additional 80 weeks. METHODS: HAUSER-OLE was an 80-week, single-arm, open-label extension of HAUSER-RCT, a randomised controlled trial, and was conducted at 46 centres in 23 countries. Paediatric patients aged 10-17 years with heterozygous familial hypercholesterolaemia who completed 24 weeks of monthly treatment with subcutaneously administered placebo or 420 mg evolocumab in HAUSER-RCT with no serious treatment-emergent adverse events were eligible to enrol in HAUSER-OLE. All patients received open-label subcutaneous evolocumab 420 mg monthly with background statins with or without ezetimibe for 80 additional weeks. The primary endpoint was treatment-emergent adverse events. Efficacy was evaluated by changes in lipids from the baseline of HAUSER-RCT to the end of HAUSER-OLE (104 weeks). This study is registered with ClinicalTrials.gov (NCT02624869) and is now completed. FINDINGS: Between Sept 10, 2016, and Nov 25, 2019, 157 patients were enrolled in HAUSER-RCT and received randomised treatment; 150 continued to HAUSER-OLE, received evolocumab treatment, and were included in the full analysis set, presented here. 146 (97%) of 150 patients completed the open-label extension. The incidence of treatment-emergent adverse events in HAUSER-OLE was 70% (105 of 150). Overall, the most common treatment-emergent adverse events were nasopharyngitis (22 [15%] of 150), headache (14 [9%]), and influenza-like illness (13 [9%]). Serious treatment-emergent adverse events occurred in four (3%) of 150 patients (perforated appendicitis and peritonitis, wrist fracture, anorexia nervosa, and headache); none was considered related to evolocumab. No treatment-emergent adverse events led to treatment discontinuation. At week 80, the mean percentage change from baseline in LDL cholesterol was -35·3% (SD 28·0). INTERPRETATION: After 80 weeks of treatment, evolocumab was safe, well tolerated, and led to sustained reductions in LDL cholesterol in paediatric patients with heterozygous familial hypercholesterolaemia. When lipid goals cannot be achieved with conventional treatments, evolocumab is an effective add-on therapy in paediatric patients. FUNDING: Amgen. TRANSLATIONS: For the French, Spanish, Spanish, Portuguese, Italian and Dutch translations of the abstract see Supplementary Materials section.

Proprotein convertase subtilisin/kexin type 9 inhibition after acute coronary syndrome or prior myocardial infarction.

PURPOSE OF REVIEW: Lowering low-density lipoprotein cholesterol (LDL-C) with statins or ezetimibe reduces major adverse cardiovascular events (MACE) in patients with coronary heart disease. Additional treatment with proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors may lower LDL-C to levels not achievable with conventional lipid-lowering agents. This review summarizes findings from two large, placebo-controlled trials that evaluated the cardiovascular efficacy of monoclonal antibodies directed against PCSK9, added to background statin therapy, in patients with established atherosclerotic cardiovascular disease (ASCVD) or recent acute coronary syndrome (ACS) and persistent elevation of atherogenic lipoproteins despite statin treatment. RECENT FINDINGS: The FOURIER trial with evolocumab and the ODYSSEY OUTCOMES trial with alirocumab demonstrated 15% overall reductions in MACE compared to placebo, associated with average achieved LDL-C levels as low as 30-40 mg/dl. Alirocumab treatment was associated with fewer deaths after ACS. Subgroups with large absolute treatment benefit included those with baseline LDL-C ≥100 mg/dl, diabetes, polyvascular or peripheral artery disease, prior coronary bypass surgery, statin intolerance, or elevated lipoprotein(a) levels. No safety concerns arose with use of PCSK9 monoclonal antibodies, even in patients who achieved LDL-C levels below 20 mg/dl. SUMMARY: In selected patients with established ASCVD or recent ACS, PCSK9 inhibitors can play an important role in reducing the risk of MACE, and may also reduce the risk of death after ACS.

Data Recorded in Real Life Support the Safety of Nattokinase in Patients with Vascular Diseases.

Nattokinase (NK) is a serine protease enzyme with fibrinolytic activity. Even if it could be used for the treatment of several diseases, no data have been published supporting its use patients who underwent vascular surgery. In this study, we evaluated both the efficacy and the safety of nattokinase (100 mg/day per os) in patients admitted to vascular surgery. Patients were of both sexes, >18 years of age, with vascular diseases (i.e., deep vein thrombosis, superficial vein thrombosis, venous insufficiency), and naïve to specific pharmacological treatments (anticoagulants or anti-platelets). Patients were divided into three groups. Group 1: patients with deep vein thrombosis, treated with fondaparinux plus nattokinase. Group 2: patients with phlebitis, treated with enoxaparin plus nattokinase. Group 3: patients with venous insufficiency after classical surgery, treated with nattokinase one day later. During the study, we enrolled 153 patients (age 22-92 years), 92 females (60.1%) and 61 males (39.9%;), and documented that nattokinase was able to improve the clinical symptoms (p < 0.01) without the development of adverse drug reactions or drug interactions. Among the enrolled patients, during follow-up, we did not record new cases of vascular diseases. Attention to patients' clinical evolution, monitoring of the INR, and timely and frequent adjustment of dosages represent the cornerstones of the safety of care for patients administered fibrinolytic drugs as a single treatment or in pharmacological combination. Therefore, we can conclude that the use of nattokinase represents an efficient and safe treatment able to both prevent and treat patients with vascular diseases.

Real-World Efficacy of Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitors (PCSK9i) in Heterozygous Familial Hypercholesterolemia Patients Referred for Lipoprotein Apheresis.

BACKGROUND A small proportion of familial hypercholesterolemia (FH) patients can adequately control this condition, although achieving the recommended targets for low-density lipoprotein cholesterol (LDL-c) levels remains a challenge. Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) are new and potent lipid-lowering drugs. However, there is scarce literature on real-world data about their use in patients with FH. MATERIAL AND METHODS We examined the reduction in LDL-c levels from the baseline, after PCSK9i initiation in heterozygous familial hypercholesterolemia patients referred for lipoprotein apheresis in our regional lipid clinic. The study was conducted from March 2018 to September 2019, the period immediately after PCSK9i reimbursement was available in France. PCSK9i was added on top of the patients' maximal tolerated lipid-lowering regimens. RESULTS The study had 123 patients with heterozygous FH. The mean age of the patients was 59±11 years. The mean baseline LDL-c for all the participants was 277±78 mg/dl. It was 283±81 mg/dl in the PCSK9i monotherapy group (n=83), 247±68 mg/dl in the PCSK9i plus ezetimibe group (n=12), and 264±78 mg/dl in the PCSK9i plus statin and ezetimibe group (n=28). The mean decrease observed in the LDL-c level from baseline was 136±70 mg/dl (n=123), 125±60 mg/dl (n=83), 103±77 mg/dl (n=12), and 175±70 mg/dl (n=28), respectively. CONCLUSIONS An overall reduction of 49.1% from the baseline LDL-c was observed in the heterozygous FH population after PCSK9i initiation in a real-world experience. The group treated with PCSK9i ezetimibe plus statin showed further reduction of their LDL-c levels with a better responder rate, achieving the target 50% reduction in LDL-c from the baseline.

Nattokinase Attenuates Retinal Neovascularization Via Modulation of Nrf2/HO-1 and Glial Activation.

Purpose: Nattokinase (NK), an active ingredient extracted from traditional food Natto, has been studied for prevention and treatment of cardiovascular diseases due to various vasoprotective effects, including fibrinolytic, antihypertensive, anti-atherosclerotic, antiplatelet, and anti-inflammatory activities. Here, we reported an antineovascular effect of NK against experimental retinal neovascularization. Methods: The inhibitory effect of NK against retinal neovascularization was evaluated using an oxygen-induced retinopathy murine model. Expressions of Nrf2/HO-1 signaling and glial activation in the NK-treated retinae were measured. We also investigated cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) after NK administration. Results: NK treatment significantly attenuated retinal neovascularization in the OIR retinae. Consistently, NK suppressed VEGF-induced cell proliferation and migration in a concentration-dependent manner in cultured vascular endothelial cells. NK ameliorated ischemic retinopathy partially via activating Nrf2/HO-1. In addition, NK orchestrated reactive gliosis and promoted microglial activation toward a reparative phenotype in ischemic retina. Treatment of NK exhibited no cell toxicity or anti-angiogenic effects in the normal retina. Conclusions: Our results revealed the anti-angiogenic effect of NK against retinal neovascularization via modulating Nrf2/HO-1, glial activation and neuroinflammation, suggesting a promising alternative treatment strategy for retinal neovascularization.

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.

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.

Serrapeptase and nattokinase intervention for relieving Alzheimer's disease pathophysiology in rat model.

Serrapeptase (SP) and nattokinase (NK) are proteolytic enzymes belonging to serine proteases. In this study, we hypothesized that SP and NK could modulate certain factors that are associated with Alzheimer's disease (AD) pathophysiology in the experimental model. Oral administration of aluminium chloride (AlCl3) in a dose of 17 mg/kg body weight (bw) daily for 45 days induced AD-like pathology in male rats with a significant increase in brain acetylcholinesterase (AchE) activity, transforming growth factor ß (TGF-ß), Fas and interleukin-6 (IL-6) levels. Meanwhile, AlCl3 supplementation produced significant decrease in brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) when compared with control values. Also, AlCl3 administration caused significant decline in the expression levels of disintegrin and metalloproteinase domain 9 (ADAM9) and a disintegrin and metalloproteinase domain 10 (ADAM10) genes in the brain. Histological investigation of brain tissue of rat model of AD showed neuronal degeneration in the hippocampus and focal hyalinosis with cellular as well as a cellular amyloid plaques formation. Oral administration of SP or NK in a rat model of AD daily for 45 days resulted in a significant decrease in brain AchE activity, TGF-ß, Fas and IL-6 levels. Also, the treatment with these enzymes produced significant increase in BDNF and IGF-1 levels when compared with the untreated AD-induced rats. Moreover, both SP and NK could markedly increase the expression levels of ADAM9 and ADAM10 genes in the brain tissue of the treated rats. These findings were well confirmed by the histological examination of the brain tissue of the treated rats. The present results support our hypothesis that the oral administration of proteolytitc enzymes, SP and/or NK, would have an effective role in modulating certain factors characterizing AD. Thus, these enzymes may have a therapeutic application in the treatment of AD.

Management of amputation-stump breakdown.

Seven cases of above-knee amputation stump breakdown following surgery for arteriosclerosis obliterans were successfully treated with Travase ointment. (Bacillus subtilis proteinase). Rehabilitation was facilitated because secondary closure of the previously infected necrotic wounds could be more rapidly undertaken.

Proteolytic enzymes as adjuncts to antimicrobial prophylaxis of contaminated wounds.

When a tissue is injured, its vessels exhibit a marked increase in vascular permeability. Blood proteins, including fibrinogen, traverse the vessel walls and lead to the development of a surface coagulum. This inflammatory response continues until primary closure of the wound edges is accomplished. The thickness of the surface coagulum is roughly proportional to the time interval between wounding and closure. This coagulum encompasses the surface contaminants, preventing contact with either topical or systemic antibiotics. The presence of this surface coagulum limits the time in which antibiotic prophylaxis is effective. At three hours after injury, antimicrobial prophylaxis of contaminated wounds has no therapeutic value. Hydrolysis of the protein coagulum by proteolytic enzymes enhances the activity of the antibiotic in experimental wounds. The success of proteolytic enzymes as adjuncts to delayed antibiotic treatment can be correlated with the clot lysis activity of the enzymes in vitro. Travase, the most potent fibrinolytic enzyme, is the most effective adjunct to delayed antibiotic therapy of contaminated wounds. In contrast, the active enzymes found in Elase, which exhibit no significant clot lysis activity in vitro, do not potentiate the activity of antibiotics in wounds subjected to a delay in treatment. Travase prolongs the period of effective topical antibiotic action for at least eight hours in experimental contaminated wounds. The therapeutic merit of Travase is also apparent when the antibiotic is administered systemically. Travase shows promise as an adjunct to a variety of antibiotics that are effective against both gram-positive and gram-negative organisms. The results of these experimental studies support our belief that clinical studies support our belief that clinical studies should now be initiated to test the therapeutic value of Travase as an adjunct to antibiotics in heavily contaminated wounds subjected to an unavoidable delay in treatment.