The clinical pharmacology of cyclooxygenase-2-selective and dual inhibitors.

Over the past decade, there have been several nonsteroidal anti-inflammatory drugs (NSAIDS) introduced in veterinary medicine with an increased gastrointestinal safety profile consistent with a cyclooxygenase (COX)-1-sparing effect. More recently, an NSAID with additional 5-lipoxygenase (5-LOX) activity has also been approved for use. Although it is tempting to equate in vitro COX-2/COX-1 and 5-LOX inhibition to overall in vivo safety, the data do not support this approach. The true overall safety for any individual compound is based on its evaluation in laboratory margin-of-safety studies, reproductive safety studies, and blind multicenter field studies in client-owned animals. Therefore, when choosing a COX-2-selective or dual-inhibitor NSAID for clinical use, all in vivo data must be taken into account to understand comparative safety, and continued use must be based on the drug's performance in the individual being treated. Until head-to-head trials in multicenter blind studies are published, comments on comparative safety and effectiveness must be reserved.     

旋毛虫Kazal型丝氨酸蛋白酶抑制剂的表达及免疫相关性

根据GenBank上旋毛虫Kazal型丝氨酸蛋白酶抑制剂(KaSPI)基因编码序列设计特异性引物,进行RT-PCR扩增,将所获PCR产物克隆至pEASY-T1载体后转入克隆感受态Tans5α,经过PCR和EcoRⅠ、XhoⅠ双酶切鉴定后进行测序。将所获目的基因与载体pET-30a(+)相连接,然后将鉴定正确的重组表达质粒pET-TsKaSPI转化到大肠杆菌BL21(DE3)中,利用IPTG诱导蛋白表达。SDS-PAGE电泳分析所得融合蛋白大小约为38 000,与预测蛋白理论值大小相符,主要以包涵体形式存在。对纯化后的重组蛋白进行Western blot鉴定,结果显示该蛋白可以被感染旋毛虫小鼠阳性血清所识别,具有良好的抗原性。将纯化的重组蛋白经腹腔注射到小鼠体内检测其对小鼠的免疫保护性,结果表明,旋毛虫肌幼虫减虫率为38.3%。通过间接ELISA法检测血清抗体水平,结果显示重组蛋白免疫小鼠血清的抗重组蛋白抗体IgG总体水平显著高于感染对照组和佐剂组。     

Irradiation of chicken egg white. I. Effect on proteinase inhibitors

Commercial ovomucoid trypsin inhibitors (OMTI) and egg white were exposed to various doses of gamma irradiation, using a cobalt-60 source. The inhibitory activity of non-irradiated and irradiated samples on bovine trypsin was tested by the Kunitz method (Kunitz 1947). The activity of the inhibitors was shown to decrease exponentially with increasing radiation dose. The D₁₀ for OMTI was 0.6 Mrad and for the trypsin inhibitors in the crude egg white diluted 1: 5 in saline 3.0 Mrad under the conditions used. The activity of the non-irradiated and irradiated inhibitors upon various animal, microbial and plant proteinases was also determined by using the crosswise casein precipitating inhibition test (crosswise CPI-test) (Fossum 1970a). By this method, which is more sensitive than the Kunitz method, trypsin inhibitory activity in egg white could be detected after exposure to an irradiation dose of 9 Mrad. No inhibitory activity could be found against any enzyme after an irradiation dose of 15 Mrad.Irradiation as a preservation method for egg and egg products is discussed.     

Immunoglobulin concentrations and bovine enterovirus inhibitors in fetal bovine fluids.

Fluids from 53 bovine fetuses ranging in age from 90 to 240 days were examined for immunoglobulin G (IgG) and immunoglobulin M (IgM) and neutralizing activity to ten bovine viruses. Non-specific inhibitors to bovine enteroviruses were found in serum, allantoic, and amniotic fluids of most samples tested. In most cases, serum IgG were within normal values. Neither IgG nor IgM was detected in amniotic fluids, whereas 2 samples of allantoic fluid contained traces of IgG.     

Heat stable inhibitors produced in raw milk.

Twenty raw farm milk samples were tested for inhibitory substances before and after being kept at room temperature until the pH fell to less than 6.3 but greater than 6.2. The tests were done using the IDF-method and the commercial Thermocult method. In both methods B. stearothermophilus var. chalidolactis is used as the test organism. In two instances a thermostable inhibitor was produced with an inhibitory potential greater than 0.01 IU/ml penicillin standard. The IDF-method was found unsuitable for the study of relatively small concentrations of antibiotics or other chemotherapeutics in milk.     

Ocular hypotensive effects of carbonic anhydrase inhibitors in normotensive and glaucomatous Beagles.

Four carbonic anhydrase inhibitors (acetazolamide, dichlorphenamide, ethoxzolamide, and methazolamide) cause ocular hypotony in normotensive and glaucomatous Beagles. Four dosages of acetazolamide and methazolamide and three dosages of dichlorphenamide and ethoxzolamide were evaluated. The extent of ocular hypotony after these carbonic anhydrase inhibitors was usually greater in glaucomatous Beagles than it was in normotensive Beagles.     

The role of small molecule inhibitors for veterinary patients.

Advances in molecular biology over the past several years have permitted a much more detailed understanding of cellular dysfunction at the biochemical level in cancer cells. This has resulted in the identification of novel targets for therapeutic intervention, including proteins that regulate signal transduction, gene expression, and protein turnover. In many instances, small molecules are used to disrupt the function of these targets, often through competitive inhibition of ATP binding or the prevention of necessary protein-protein interactions. Future challenges lie in identifying appropriate targets for intervention and combining small molecule inhibitors with standard treatment modalities, such as radiation therapy and chemotherapy.     

Conservation of nitrogen in cattle feedlot waste with urease inhibitors.

Feedlot cattle normally retain less than 20% of their dietary nitrogen intake. Sixty to 80% of the nitrogen excreted is normally lost through volatilization of ammonia, which is primarily generated from urea. This loss of ammonia nitrogen pollutes the environment and creates an unfavorable ratio of nitrogen to phosphorous (N:P) in the waste for crop growth. Two urease inhibitors, cyclohexylphosphoric triamide (CHPT) and N-(n-butyl) thiophosphoric triamide (NBPT) were evaluated for their ability to reduce the rate of urea hydrolysis in beef cattle feedlot pens. Initially, a total of six pens were used, two pens per treatment, with approximately 70 cattle per pen, and a single topical application of CHPT or NBPT at 20 mg/kg of manure. Essentially no urea was found in untreated pens. However, with CHPT treatment, 2 g of urea/kg of dry manure accumulated by d 4, and all gradually disappeared by d 11; NBPT conserved 3 and 3.5 g of urea/kg by d 4 and 9, respectively, and it had disappeared by d 14 (treatment [trt] x day, P = .003). A second study involved application of NBPT weekly for 6 wk. This caused urea to accumulate to a peak concentration of 17 g/kg of manure by d 30 (trt x day2, P = .001). Once the treatment was stopped the urea concentration began to decrease. When the NBPT was applied weekly, the concentration of ammonia in the waste was less for the treated pens (trt x day, P = .01), the total nitrogen was greater (trt x day, P = .04), pH tended to be lower (trt x day, P = .10), and the total volatile acids were not different (trt x day, P = .51) from untreated pens. We concluded that urease inhibitors could be used to control ammonia emissions from animal wastes, prevent environmental damage, and produce a more balanced (N:P) fertilizer from manure.     

Motor unit irritability in Beagles Before and after exposure to cholinesterase inhibitors.

Electromyographic (EMG) examinations were performed on Beagles before and for 7 days after oral administration of one of the following organophosphate (OP) compounds; ronnel (55.0 or 110.0 mg/kg), dichlorvos (29.7, 59.4, or 148.5 mg/kg), or cythioate (24.8 or 33.0 mg/kg). The EMG values determined were evoked potentials, after-discharge activity, F-wave activity, nerve conduction velocity, and motor unit potential activity associated with interosseous and pectineal reflexes. Erythrocyte cholinesterase (ChE) activities were measured in some dogs. Ronnel did not have an effect on ChE activity, whereas dichlorvos and cythioate, at all dosage levels, had an inhibitory effect. Some dogs had minor signs of OP toxicosis. The EMG changes for individual OP compounds were not statistically significant (P greater than 0.05), but pooled results revealed an increased duration of evoked potentials, increased after-discharge activity, and decreased F-wave activity; however, only the effect on duration was significant (P less than 0.05). Reflex motor unit potential activity and nerve conduction velocities were not affected. Effects of neostigmine (0.1 to 0.4 mg/kg) given IV to anesthetized, atropinized Beagles were similar to those effects shown by pooled data for the OP compounds, but considerably more muscle fasciculation was produced. Results of this study indicate that even when erythrocyte ChE activity is reduced by OP compounds at dosage levels that produce no or minimal visible signs of toxicosis, EMG reveals little evidence for increased motor unit irritability.     

Kinase dysfunction and kinase inhibitors

With recent advances in molecular biology, abnormalities in cancer cells that contribute to dysregulation of cell survival and proliferation are being characterized with greater precision. Through this process, key abnormalities in cancer cells involving proteins that regulate signal transduction, migration, mitosis and other critical processes have been identified. Such abnormalities often involve a class of proteins called kinases that act to phosphorylate other proteins in the cell, resulting in activation of these proteins in the absence of appropriate stimulation/regulation. Given their role in tumour biology, substantial effort has been directed at blocking the function of these proteins. Several approaches have been used, including monoclonal antibodies and small molecule inhibitors. While antibodies are primarily directed at cell surface proteins, small molecule inhibitors, also known as kinase inhibitors, target proteins throughout the cell. A variety of kinase inhibitors have been approved for the treatment of human cancers. In some instances, these inhibitors have exhibited significant clinical efficacy, and it is likely that their biological activity will be further enhanced as combination regimens with standard treatment modalities are explored. The use of kinase inhibitors in dogs and cats is relatively recent, although two inhibitors, toceranib (Palladia; Pfizer Animal Health, Madison, NJ, USA) and masitinib (Kinavet; Catalent Pharma Solutions, Somerset, NJ, USA) have been approved by the Federal Drug Administration (USA) for use in dogs. This article reviews the biology of protein kinase dysfunction in human and animal cancers, and the application of specific kinase inhibitors to veterinary cancer patients.     

Influence of cyclooxygenase inhibitors on furosemide-induced hemodynamic effects during exercise in horses.

Furosemide, which commonly is used as a prophylactic treatment for exercise-induced pulmonary hemorrhage in horses, may mediate hemodynamic changes during exercise by altering prostaglandin metabolism. To determine if furosemide's hemodynamic effects during exercise in horses could be reversed, cyclooxygenase inhibitors were administered with furosemide. Four treatments were administered 4 hours prior to treadmill exercise at 9 and 13 m/s. They included a control treatment (10 ml of 0.9% NaCl solution, IV), furosemide (1 mg/kg of body weight, IV) administered alone, and furosemide in combination with phenylbutazone (4 mg/kg, IV, q 12 h for 2 days) or with flunixin meglumine (1.1 mg/kg, IV, on the day of experiment). Five horses were randomly assigned to complete all treatments. Physiologic variables at rest prior to exercise were not influenced by treatments. Furosemide, administered alone, reduced mean right atrial pressure and mean pulmonary artery pressure during exercise. The combinations of furosemide and flunixin meglumine or furosemide and phenylbutazone, at both levels of exercise intensity, returned mean right atrial pressure and mean pulmonary artery pressure to the value of the control treatment. During rest and exercise, plasma lactate concentration, PCV, heart rate, mean carotid artery pressure, oxygen consumption, carbon dioxide elimination, and cardiac output were not altered by any of the treatments. At 5 minutes after exercise, the administration of furosemide, alone or with phenylbutazone, reduced mean right atrial pressure. Other measured variables were not significantly influenced by treatments during recovery from exercise. These results suggested that cyclooxygenase inhibition partially reverses the decrease in mean right atrial pressure or pulmonary artery pressure induced by furosemide during exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro effects of cyclooxygenase inhibitors in whole blood of horses, dogs, and cats.

OBJECTIVE: To determine potency and selectivity of nonsteroidal anti-inflammatory drugs (NSAID) and cyclooxygenase- (COX-) specific inhibitors in whole blood from horses, dogs, and cats. SAMPLE POPULATION: Blood samples from 30 healthy horses, 48 healthy dogs, and 9 healthy cats. PROCEDURE: Activities of COX-1 and COX-2 were determined by measuring coagulation-induced thromboxane and lipopolysaccharide-induced prostaglandin E2 concentrations, respectively, in whole blood with and without the addition of various concentrations of phenylbutazone, flunixin meglumine, ketoprofen, diclofenac, indomethacin, meloxicam, carprofen, 5-bromo-2[4-fluorophenyl]-3-14-methylsulfonylphenyl]-thiophene (DuP 697), 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furan one (DFU), 3-(3,4-difluorophenyl)-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone (MF-tricyclic), and celecoxib. Potency of each test compound was determined by calculating the concentration that resulted in inhibition of 50% of COX activity (IC50). Selectivity was determined by calculating the ratio of IC50 for COX-1 to IC50 for COX-2 (COX-1/COX-2 ratio). RESULTS: The novel compound DFU was the most selective COX-2 inhibitor in equine, canine, and feline blood; COX-1/COX-2 ratios were 775, 74, and 69, respectively. Carprofen was the weakest inhibitor of COX-2, compared with the other COX-2 selective inhibitors, and did not inhibit COX-2 activity in equine blood. In contrast, NSAID such as phenylbutazone and flunixin meglumine were more potent inhibitors of COX-1 than COX-2 in canine and equine blood. CONCLUSIONS AND CLINICAL RELEVANCE: The novel COX-2 inhibitor DFU was more potent and selective in canine, equine, and feline blood, compared with phenylbutazone, flunixin meglumine, and carprofen. Compounds that specifically inhibit COX-2 may result in a lower incidence of adverse effects, compared with NSAID, when administered at therapeutic dosages to horses, dogs, and cats.     

Effect of pH, temperature, and inhibitors on autolysis and catalytic activity of bovine skeletal muscle mu-calpain.

To improve our understanding of the regulation of calpain activity in situ during postmortem storage, the effects of pH, temperature, and inhibitors on the autolysis and subsequent proteolytic activity of mu-calpain were studied. Calpains (mu- and m-calpain) and calpastatin were purified from bovine skeletal muscle. All autolysis experiments were conducted in the absence of substrate at different pH (7.0, 6.2, and 5.8) and temperatures (25 and 5 degrees C). Autolysis of mu-calpain generated polypeptides with estimated masses of 61, 55, 40, 27, 23, and 18 kDa. The rate of autolysis was significantly increased with decreasing pH. The rate of degradation of the 80-kDa subunit was significantly decreased with decreasing temperature. However, degradation of the 30-kDa subunit was not affected by decreasing temperature. By conducting autolysis experiments at 5 degrees C and immunoblotting of autolytic fragments with anti-80 kDa, it was demonstrated that with the exception of 18 kDa, which originates from 30 kDa, all other fragments probably originate from degradation of the 80-kDa subunit. Calpastatin, leupeptin, and E-64 did not inhibit the initial step of autolysis, but they did inhibit further breakdown of these fragments. However, zinc, which also inhibits the proteolytic activity of calpain, only reduced the rate of autolysis, but did not inhibit it. The possible significance of these results in terms of the regulation of calpain in postmortem muscle is discussed.     

Effect of endocytic and metabolic inhibitors on the internalization and intracellular growth of Brucella abortus in Vero cells.

Uptake, transfer to rough endoplasmic reticulum, and intracellular growth of Brucella abortus were studied in Vero cells treated with endocytic and metabolic inhibitors. Infection of Vero cells was suppressed when inhibitors of energy metabolism (iodoacetate, dinitrophenol), receptor-mediated endocytosis (monodansylcadaverine, amantadine, methylamine), or endosomal acidification (chloroquine, ammonium chloride, monensin) were added to the inoculum. Inhibition was not observed when these drugs were added after the inoculation period. Infection of Vero cells by B abortus was inhibited by dibutyryl-cyclic adenosine monophosphate and Vibrio cholerae enterotoxin, but was stimulated by dibutyryl-cyclic guanosine monophosphate and escherichia coli heat-stable enterotoxin a. Uptake of B abortus by Vero cells was not prevented by colchicine, but was abolished by cytochalasin B. Uptake of heat-killed B abortus and noninvasive E coli was similar to that of viable brucellae. Intracellular growth of B abortus was not affected by cycloheximide. Results indicate that: B abortus may be internalized by a receptor-mediated phagocytic process; transfer of B abortus from phagosomes to rough endoplasmic reticulum may require endosomal acidification; and replication of B abortus within the rough endoplasmic reticulum may not depend on protein synthesis by the host cell.     

β-内酰胺酶抑制剂的研究进展

β-内酰胺酶抑制剂在对抗β-内酰胺酶耐药菌感染中发挥着重要作用,一直是药物化学领域的研究热点,但β-内酰胺酶变异体多元化及不利突变减缓了新型抑制剂的发展进程。诸多国内外相关文献在深入了解β-内酰胺酶水解机制及活性位点关键氨基酸作用特点的基础上,通过对已有化合物进行结构修饰或基于片段设计来筛选新的β-内酰胺酶抑制剂。详细介绍了β-内酰胺酶抑制剂的研究进展,旨在为进一步深入开展以增强化合物与酶关键氨基酸互作为基础的创新药物筛选提供帮助。     

Mode of action of proteinase inhibitors in rats. 2. Effect of leupeptin on N-balance and biological half-life in ad lib feeding

During a 15-day period, growing Wistar rats (75 g) were fed ad lib. a whole-egg diet containing 10% crude protein (control group) and the same diet + a daily subcutaneous injection of 1 mg of leupeptin per animal (experimental group), respectively. To investigate the influence of the peptide aldehyde leupeptin on the M-metabolism, N-balance trials were carried out between the 1st and the 6th, and the 10th and the 15th experimental days. Leupeptine was not found to influence the true N-digestibility. However, the intermediary N-utilization, characterized by the biological value of the dietary protein, deteriorated in comparison with the control group. The peptide aldehyde under study resulted in an extension of the biological half-life of the proteins in the tissue of the small and large intestine. It was measured via the decline of radioactivity in protein following application of L-guanidino-14 C-arginine and L-(4,5-3H)-leucine. The amino acid utilization appears to have been increased as well. In connection with previous studies (SIMON et al., 1976) i, which, under somewhat different experimental conditions, leupeptine resulted in a higher intermediary N-utilization, the mentioned agent is supposed to be able to influence N-utilization. However, additional studies are required to make statements regarding the influence of dosis and metabolic situation.