Enhanced Bioavailability of Subcutaneously Injected Insulin by Pretreatment with Ointment Containing Protease Inhibitors

The present study was undertaken to develop an ointment preparation containing a protease inhibitor for stabilizing subcutaneously injected insulin. The ointment containing the protease inhibitor, gabexate mesilate or nafamostat mesilate, was applied to the skin around the insulin injection site. Three results were obtained. First, gabexate and nafamostat inhibited insulin degradation in subcutaneous tissue homogenates in vitro. Second, after application of gabexate or nafamostat ointment, an appreciable amount of gabexate or nafamostat appeared in the subcutaneous tissue of rats or hairless mice and their concentrations were comparable to those seen in the in vitro experiment. Third, insulin degradation at the subcutaneous injection site in the rat was depressed after pretreatment with gabexate or nafamostat ointment. Pretreatment with gabexate or nafamostat ointment increased the plasma immunoreactive insulin (IRI) levels and the hypoglycemic effect of insulin in healthy volunteers. These results indicate that gabexate or nafamostat ointments stabilize subcutaneously injected insulin.     

Changes in blood viscosity with synthetic protease inhibitors

We examined the effects on whole blood viscosity and coagulation time of various dosages of the synthetic low-molecular protease inhibitors gabexate mesilate and nafamostat mesilate with an oscillation-type viscometer. When either agent was added, blood viscosity decreased dose-dependently along a sigmoid-like curve. Furthermore, coagulation time was shorter with gabexate mesilate than with nafamostat mesilate owing to the differences of half-life in human blood. Thrombin generation, which results from the activation of coagulation factors, is inhibited by synthetic protease inhibitors and subsequently decreases blood viscosity dose-dependently.     

Nafamostat mesilate is an extremely potent inhibitor of human tryptase

Previously, nafamostat mesilate was found to be a potent inhibitor of human tryptase. In present study, we performed a kinetic study to determine its K(i) value for tryptase and compared it with that of gabexate mesilate. Nafamostat mesilate inhibited human tryptase in a competitive manner. The apparent K(i) value was estimated to be 95.3 pM, which was 1000 times lower than that of gabexate mesilate (95.1 nM). These results strongly indicated that nafamostat mesilate is an extremely potent inhibitor of tryptase and suggested that some of its beneficial effects in the treatment of clinical status may be due to tryptase inhibition.     

A potent tryptase inhibitor nafamostat mesilate dramatically suppressed pulmonary dysfunction induced in rats by a radiographic contrast medium

(1) Intravenous injection of ioxaglate (4 g iodine kg(-1)), an iodinated radiographic contrast medium, caused a marked protein extravasation, pulmonary oedema and a decrease in the arterial partial oxygen pressure in rats. (2) All of these reactions to ioxaglate were reversed by the pretreatment with gabexate mesilate (10 and 50 mg kg(-1), 5 min prior to injection) or nafamostat mesilate (3 and 10 mg kg(-1)), in which the inhibition was complete after injection of nafamostat mesilate (10 mg kg(-1)). (3) Both gabexate mesilate and nafamostat mesilate inhibited the activity of purified human lung tryptase, although the latter compound was far more potent than the former. (4) Ioxaglate enhanced the nafamostat-sensitive protease activity in the extracellular fluid of rat peritoneal mast cell suspensions. (5) Tryptase enhanced the permeability of protein through the monolayer of cultured human pulmonary arterial endothelial cells. Ioxaglate, when applied in combination with rat peritoneal mast cells, also produced the endothelial barrier dysfunction. These effects of tryptase and ioxaglate were reversed by nafamostat mesilate. (6) Consistent with these findings, immunofluorescence morphological analysis revealed that tryptase or ioxaglate in combination with mast cells increased actin stress fibre formation while decreasing VE-cadherin immunoreactivity. Both of these actions of tryptase and ioxaglate were reversed by nafamostat mesilate. (7) These findings suggest that tryptase liberated from mast cells plays a crucial role in the ioxaglate-induced pulmonary dysfunction. In this respect, nafamostat mesilate may become a useful agent for the cure or prevention of severe adverse reactions to radiographic contrast media.     

Effect of exogenous epidermal growth factor (EGF) on nonenzymatic antioxidant capacities and MPO activity of wound tissue

Growth factors and cytokines are key regulators of the wound-healing process. Epidermal growth factor (EGF) plays a vital role during the process of wound healing. There are limited numbers of studies related to EGF implantation effects on oxidative stress in oral wound healing. The objective of this study was to investigate the effect of EGF on myeloperoxidase (MPO) activity and nonenzymatic antioxidant levels of oral wound tissue on different days. In this study, New Zealand male albino rabbits were used. After submucosal incisions, the rabbits were divided into two equal groups: untreated wounds, and EGF-implanted wounds. The levels of glutathione (GSH), ascorbic acid (AA), and MPO activity were measured spectrophotometrically. As a result, MPO activity of oral wound tissue strips was increased by exogenous EGF treatment compared with controls. In addition, GSH and AA levels of oral wound tissue strips were not changed during oral wound-healing phases for both controls and experimental groups.     

Additional mechanisms of nafamostat mesilate-associated hyperkalaemia

Objective: Nafamostat mesilate, a potent protease inhibitor, is widely used for the treatment of pancreatitis, disseminated intravascular coagulation and as an anticoagulant in haemodialysis. However, hyperkalaemia associated with nafamostat mesilate has been reported. It is thought to be due to decreased urinary potassium excretion, of the drug suppression of aldosterone secretion, and a direct inhibitory action on the apical Na⁺ conductance in collecting ducts. We have seen two cases of nafamostat mesilate associated-hyperkalaemia, which indicated that extrarenal potassium imbalance might play a role in inducing hyperkalaemia. Methods: To examine the effect of nafamostat mesilate on potassium transport in erythrocytes in vitro, ⁸⁶RbCl uptake was measured in red blood cells from eight healthy volunteers. Results: Nafamostat mesilate and a metabolite, 6-amidino-2-naphthol, at concentrations of 10⁻⁴ and 10⁻³M, respectively, significantly, suppressed potassium influx whilst another metabolite, p-guanidinobenzoic acid, had no effect. The inhibitory action of nafamostat mesilate was not affected by various inhibitors. Conclusion: Nafamostat mesilate and its metabolite, 6-amidino-2-naphthol, suppressed potassium influx in erythrocytes by inhibition of a Na-K ATPase dependent pathway, which was not inhibited by amiloride, barium, nor by frusemide (furosemide). Received: 15 January 1996/Accepted in revised form: 7 May 1996     

Improving effect of the synthetic protease inhibitor E-3123 on experimental DIC in dogs]

Disseminated intravascular coagulation (DIC) is a severe syndrome associated with generalized, intractable bleeding and multiple organ failure. Synthesized protease inhibitors such as gabexate mesilate and nafamostat mesilate show an improving effect on DIC, which develops by a chain reaction involving the coagulation, fibrinolysis, complement and kallikrein systems. Experimental DIC was developed in Beagle dogs by infusion of 150 U/kg tissue thromboplastin (Group I), and the improving effect of a new synthetic protease inhibitor, E-3123, was examined. The following groups of animals were treated with drugs: Group II (n = 4) was given with 5 mg/kg/hr of E-3123; group III (n = 4) was given 10 mg/kg/hr of E-3123; and group IV was given 6 mg/kg/hr of gabexate mesilate (GM). Although improvement of the hemodynamics or peripheral circulation was not apparent, a slight, but insignificant, improvement of lactate/pyruvate was noted in the treated groups. On the other hand, the hemostatic abnormalities such as prolongation of prothrombin time and activated thromboplastin time; decreases of platelet count, fibrinogen and alpha 2-antiplasmin; and increases of fibrin degradation products were significantly improved in the treated groups. These results indicate that E-3123 is effective for improving experimental DIC, and it is suggested that E-3123 is applicable for the treatment of clinical DIC.     

Comparative studies of nafamostat mesilate and various serine protease inhibitors in vitro

Inhibitory effects of nafamostat mesilate (nafamostat) on various enzymes were investigated, and they were compared with those of gabexate mesilate (gabexate), leupeptin, aprotinin and urinastatin in vitro. Nafamostat inhibited trypsin, plasmin, thrombin, pancreatic kallikrein, Clr and Cls more potently than gabexate and leupeptin. Gabexate and leupeptin did not inhibit pancreatic kallikrein and thrombin, respectively. Aprotinin inhibited trypsin, plasmin, pancreatic kallikrein and chymotrypsin. Urinastatin inhibited trypsin and chymotrypsin. Nafamostat inhibited the complement-mediated hemolysis in diluted serum more potently than gabexate and leupeptin, but aprotinin and urinastatin did not. Nafamostat, furthermore, inhibited the complement-mediated hemolysis in undiluted serum, but gabexate did not. Unlike aprotinin and urinastatin, nafamostat and gabexate inhibited alpha 2-macroglobulin bound trypsin as well as free trypsin to the same extent. The inhibitory effect of gabexate toward trypsin was reduced more markedly than that of nafamostat after incubation with plasma at 37 degrees C. These results show that nafamostat is more useful than other inhibitors such as gabexate, leupeptin, aprotinin and urinastatin.     

Effects of Epidermal Growth Factor Dosage Forms on Mice Full-thickness Skin Wound Zinc Levels and Relation to Wound Strength

Epidermal growth factor (EGF) and zinc promote re-epithelization and reparative tissue strength by enhancing deposition of collagen at the site of the wound. In this study two EGF dosage forms were chosen to assess the effect of zinc levels on wound healing and for comparison with wound tear strengths. A solution of EGF in 0.9% w/v NaCl and an EGF gel in 0.2% Carbopol 940 polymer (5 μL) were applied to full-thickness skin wounds of mice twice a day for 7 and 15 days. Wound zinc levels were higher on day 7 than on day 15, especially in wounds treated with EGF. The wound zinc levels of the gel + EGF group on day 15 were similar to those of normal control skin. These results imply that there is a close connection, but no direct relationship, between EGF application in both dosage forms and wound zinc levels during healing.     

Prevention of the adverse effects of aprotinin in autologous fibrin glue

To avoid the adverse effects of aprotinin (Apr) in autologous fibrin glue, we compared the inhibitory properties of four commercial anti-fibrinolytic agents (tranexamic acid (Tra), epsilon-aminocaproic acid (Ips), gabexate mesilate (Gab) and nafamostat mesilate (Naf)). The optimum conditions for the lysing of fibrin glue were an incubation temperature of 37 degrees C, and incubation medium containing urokinase at 100 U/ml and plasminogen at 100 mU/ml (for the washed glue), or neither (for unwashed glue). Fibrin glues without an anti-fibrinolytic agent were quickly lysed in the incubation medium, while those with an anti-fibrinolytic agent were slowly lysed dose-dependently. Naf was the most potent inhibitor and had high affinity for the glue, but the other agents were poor inhibitors and had low affinity. The inhibition potency (IP) of each agent did not correlate with hydrophobicity, but a good correlation was obtained between the remaining coefficient (RC) and hydrophobicity. Naf did not affect the adhesive strength of the glue. These results indicate that Naf is the most suitable anti-fibrinolytic agent to replace Apr.     

Nafamostat mesilate suppresses NF-kappaB activation and NO overproduction in LPS-treated macrophages

Nafamostat mesilate (NM), a clinically used serine protease inhibitor, suppressed the overproduction of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) in RAW264.7 murine macrophages treated with lipopolysaccharide (LPS, 100 ng/ml); however, it had little effect on endothelial NOS (eNOS) in human umbilical vein endothelial cells (HUVEC). Electrophoretic mobility shift assay (EMSA) revealed that LPS activated nuclear factor-kappaB (NF-kappaB) in RAW264.7 cells and that this activation was suppressed by nafamostat mesilate. Western blotting showed that nafamostat mesilate suppressed the phosphorylation and degradation of inhibitor kappaB-alpha (IkappaB-alpha), which holds NF-kappaB in the cytoplasm in an inactivated state. Our observations suggest that nafamostat mesilate is a candidate agent for various diseases such as ischemia-reperfusion, graft rejection, inflammatory diseases, and autoimmune diseases, in which iNOS and/or NF-kappaB are upregulated.     

甲磺酸加贝酯在五种输液中的稳定性

采用紫外分光光度法考察甲磺酸加贝酯在五种输液中的配伍稳定性。葡萄糖注射液 ( 10 % ,5% )、葡萄糖氯化钠注射液、复方氯化钠注射液、氯化钠注射液在 4℃ ,2 5℃ ,37℃下测定配伍后不同时间甲磺酸加贝酯含量 ,同时观察外观并测定 p H值。结果 :7h各配伍液外观无变化 ,p H值及甲磺酸加贝酯含量无明显变化     

Growth factor regulation and manipulation in wound repair: to scar or not to scar,that is the question

The process of tissue repair following injury is in the large part mediated by secreted growth factors which, in an autocrine or paracrine fashion, stimulate immune and mesenchymal cells at the site of injury. The complex process of replacing damaged tissue with newly formed tissue involves components of the blood, coagulation, immune and mesenchymal systems as well as cytokines, chemokines, metalloproteinases and growth factors. This review will focus on growth factors as the controllers of this process and includes members of the transforming growth factor (TGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), connective tissue derived growth factor (CTGF) and insulin-like growth factor-I (IGF-I) families of growth factors. These growth factors stimulate re-epithelialisation, angiogenesis, extracellular matrix (ECM) formation and cell proliferation each of which plays a role in tissue replacement and restoration of tissue function following injury. Normal wound healing frequently involves the formation of scar tissue, including increased mesenchymal cell proliferation and excessive production of ECM proteins. While scar tissue rapidly and effectively closes wounds, it leaves visually apparent tissue structure changes and may reduce the function of the tissue leading to compromised organ function. Growth factors, the conductors of these processes, are targets for therapeutic manipulation of wound healing and scar formation. Recent patents involving growth factors may be implicated in the treatment of wound healing following tissue injury. Enhanced growth factor activity may be beneficial to increase the rate of wound healing in chronic non-healing wounds, whereas reduction of growth factor presence or activity may reduce scar formation in the skin and internal organs, which may be particularly relevant where scar formation is associated with pathologic loss of life sustaining organ function.     

Wound healing properties of Indian propolis studied on excision wound-induced rats

Context: In traditional medicine propolis is widely used for the treatment of various ailments including ulcer and wound healing. The phytochemical screening of Indian propolis indicates the presence of biologically active ingredients in appreciable amounts. In the absence of systematic evaluation of wound healing properties of Indian propolis in the literature, the present study was undertaken.

Objective: The aim of this study was to evaluate the wound healing potential of Indian propolis on excision wounds induced in experimental rats.

Materials and methods: Excision wounds were created in male Wistar rats and were treated with Indian propolis ointment (nitrofurazone was used as a reference drug - widely used for wound healing) for a period of 14 days. Control rats were treated with petroleum jelly. The parameters analyzed include wound contraction, hydroxyproline, hexosamine, uronic acid, total protein, DNA, and RNA.

Results: Topical application of propolis ointment for 14 days significantly improved the wound contraction when compared to the control group of rats. The determination of hydroxyproline, hexosamine, uronic acid, DNA, RNA and protein levels in the wound matrix revealed the pro-healing effects of propolis. The results obtained were comparable with nitrofurazone.

Discussion and conclusion: It appears that the ethanol extract of Indian propolis possesses significant pro-healing activity by accelerating the healing process at various phases of tissue repair. The presence of biologically active ingredients such as flavonoids, phenolic acids, terpenes, benzoic acids, amino acids and vitamins, etc. in Indian propolis may readily account for the observed prophylactic action of propolis in wound healing.     

甲磺酸萘莫司他中有机溶剂残留的测定

目的建立气相色谱法测定甲磺酸萘莫司他中有机溶剂残留量。方法在EC-1000(30m×0.54mm×1.2μm)毛细管柱上采用程序升温法,载气为氮气,汽化室温度200℃,检测器温度220℃,以二氧六环为内标,内标法测定甲磺酸萘莫司他中残留的甲醇、丙酮、吡啶、N,N-二甲基甲酰胺。结果在本研究色谱条件下,各溶剂及内标均能得到良好的分离度,空白无干扰;在所考察的浓度范围内线性良好,相关系数均在0.9996以上,回收率96.7%～102.0%,检测限0.15～2.8μg/mL。结论试验建立的色谱方法简便、准确,灵敏度高,适合甲磺酸萘莫司他机溶剂残留量的检测。     

四妙君逸软膏换药用于促进肛肠病术后创面愈合的研究

目的:探讨四妙君逸软膏促进创面愈合的作用及机制。方法:将60只大鼠随机分成空白基质组、重组人表皮生长因子组、龙珠软膏组及四妙君逸软膏高、中、低剂量组,每组10只,制备大鼠创伤模型。治疗从造模后d 1开始至创面完全愈合,观察术后各组大鼠创面的愈合情况,于术后d 3,d 7和d 14记录创面愈合率,在d 7和d 14取材。采用考马斯亮兰法检测大鼠肉芽组织中蛋白含量、免疫组化ABC法检测表皮生长因子受体(EGFR),最后观察创面愈合时间。结果:四妙君逸软膏能缩短创面愈合时间,提高创面愈合率及创面肉芽组织中蛋白含量和EGFR。结论:四妙君逸软膏能促进创面愈合,其机制之一是通过提高EGFR以及蛋白含量来促进创面愈合。     

Bilayer hydrogel dressing with lysozyme-enhanced photothermal therapy for biofilm eradication and accelerated chronic wound repair

Biofilms are closely associated with the tough healing and dysfunctional inflammation of chronic wounds. Photothermal therapy (PTT) emerged as a suitable alternative which could destroy the structure of biofilms with local physical heat. However, the efficacy of PTT is limited because the excessive hyperthermia could damage surrounding tissues. Besides, the difficult reserve and delivery of photothermal agents makes PTT hard to eradicate biofilms as expectation. Herein, we present a GelMA-EGF/Gelatin-MPDA-LZM bilayer hydrogel dressing to perform lysozyme-enhanced PTT for biofilms eradication and a further acceleration to the repair of chronic wounds. Gelatin was used as inner layer hydrogel to reserve lysozyme (LZM) loaded mesoporous polydopamine (MPDA) (MPDA-LZM) nanoparticles, which could rapidly liquefy while temperature rising so as to achieve a bulk release of nanoparticles. MPDA-LZM nanoparticles serve as photothermal agents with antibacterial capability, could deeply penetrate and destroy biofilms. In addition, the outer layer hydrogel consisted of gelatin methacryloyl (GelMA) and epidermal growth factor (EGF) promoted wound healing and tissue regeneration. It displayed remarkable efficacy on alleviating infection and accelerating wound healing in vivo. Overall, the innovative therapeutic strategy we came up with has significant effect on biofilms eradication and shows promising application in promoting the repair of clinical chronic wounds.     

Evaluation of wound healing potential of topical formulation of leaf juice of tridax procumbens L. In mice

Tridax procumbens L. (Compositae) is a common weed that grows in the rice fields of India. Traditionally the juice from leaves of Tridax procumbens has been used for healing dermal wound. However, in experimental studies, equivocal pro and anti-healing action of T. procumbens has been demonstrated. The present study evaluates the effect of topical ointment formulation of the leaf juice of T. procumbens using excision wound model in mice. Excision wounds (4 mm, i.d.) were inflicted on depilated back of mice. Ointment formulation of TP (50 mg of either 1 or 4 mg/g) was applied twice daily for 4 days on the dermal wound. Similarly, control group was treated with VEGF ointment (50 mg of 1 μg/g). The parameters observed were re-epithelization, vascularity, fibroblast number, collagen content. The healing exerted by TP (1 mg/g) was comparable to VEGF (1 μg/g). On the other hand, TP (4 mg/g) induced inflammation, edematous tissue and decreased vascularity. Taken together, the results imply that TP possesses dose dependent pro-healing potential, and its high dose exerts inflammatory reaction.     

The reduction in inflammation and impairment in wound healing by using strontium chloride hexahydrate

Background: Numerous growth factors, cytokine, mitogen and chemotactic factors are involved in wound healing. Even though inflammation is important for the stimulation of proliferative phase, excessive inflammation also causes impairment in wound healing. Strontium salts suppress keratinocyte-induced TNF-alpha and interleukin-1 and interleukin-6 in in vitro cultures. This study was conducted to determine the effects of administration of topical strontium chloride hexahydrate on wound healing through TNF-alpha and TGF-beta in surgical wound healing model of in-vivo rat skin.

Material and methods: Twenty-four rats were used in the study. After approximately 2?cm cutaneous–subcutaneous incision was horizontally carried out on the mid-neckline of the rats, the incision was again closed using 2.0 vicryl. The rats were assigned into three groups including eight rats in each group. Placebo emollient ointment and also the ointments, which were containing 5% and 10% strontium chloride hexahydrate and were prepared at the same base with placebo ointment, were administered to the groups by a blind executor twice a day for a week. At the end of seventh day, the rats were sacrificed and cutaneous and subcutaneous tissue of their wound site was resected for histopathological examination. Scoring of histopathological wound healing and scoring of tissue TNF-alpha and TGF-beta level with immunohistochemical staining were performed.

Results: The groups, to which both 5% and 10% strontium chloride hexahydrate was administered, had lower immunohistochemical TNF-alpha levels and histopathological wound scores compared to controls, which was statistically significant (p?Conclusion: Strontium chloride hexahydrate can lead to impairment in wound healing by suppressing inflammation through TNF-alpha.     

NAFAMOSTAT ENHANCES IBMX AND SECRETIN-STIMULATED WATER AND BICARBONATE SECRETION IN THE ISOLATED, BLOOD-PERFUSED DOG PANCREAS

1. The effect of nafamostat mesilate, a synthetic protease inhibitor, on pancreatic exocrine secretion was investigated in isolated, blood-perfused dog pancreas. 2. Intra-arterial injections of nafamostat (2–10 gmol) dosedependently enhanced water and bicarbonate secretion stimulated by 3-isobutyl-1-methylxanthine (IBMX) or secretin, although nafamostat alone did not affect the basal secretion. 3. In the nafamostat-enhanced pancreatic juice, bicarbonate concentrations were increased dose-dependently as compared with the control, while protein concentrations were not significantly changed. 4. These results indicate that nafamostat enhances IBMX and secretin-stimulated water and bicarbonate secretion, which may be mediated through CAMP activation.