Investigation of the effects of deracoxib and piroxicam on the in vitro viability of osteosarcoma cells from dogs

OBJECTIVE: To determine whether exposure of canine osteosarcoma cells to deracoxib or piroxicam results in decreased viability, whether the cytotoxic effects of deracoxib and piroxicam involve induction of apoptosis, and whether deracoxib is a more potent inhibitor of osteosarcoma cell growth than piroxicam. SAMPLE POPULATION: 1 fibroblast and 3 osteosarcoma cell lines. PROCEDURE: Cell counts and viability assays were performed using osteosarcoma cells (POS, highly metastatic POS, and canine osteosarcoma cell 31) and fibroblasts after 72 hours of incubation with deracoxib at concentrations of 0.5 microM to 500 microM or piroxicam at concentrations of 1 microM to 1,000 microM. Percentage viability was determined for each concentration. A DNA fragmentation analysis was performed to assess drug-induced apoptosis. RESULTS: Concentration of deracoxib required for 50% inhibition of cell viability (IC50) was reached in all 3 osteosarcoma cell lines and ranged from 70 to 150 microM, whereas the IC50 for piroxicam was only reached in the POS cell line at 500 microM. Neither deracoxib nor piroxicam induced sufficient toxicity in fibroblasts to reach an IC50. Exposure of osteosarcoma cells to cytotoxic concentrations of deracoxib and piroxicam did not result in DNA fragmentation. CONCLUSIONS AND CLINICAL RELEVANCE: Intermediate and high concentrations of deracoxib and high concentrations of piroxicam were cytotoxic to osteosarcoma cells; neither drug inhibited cell viability at typical plasma concentrations in dogs. Deracoxib inhibited viability of cells at concentrations that did not affect fibroblast viability. There was no evidence of apoptosis induction for either drug; however, only 1 cell line was evaluated for apoptosis induction and only for a limited selection of drug concentrations.     

Anti-elastase and anti-hyaluronidase activity of phosvitin isolated from hen egg yolk

ABSTRACT

1. Phosvitin, a major phosphoprotein found in egg yolk, has strong antioxidant activity. Activation of elastase, collagenase, and hyaluronidase by reactive oxygen species are related to the degradation of ECM and skin aging. The objective of this study was to determine the anti-elastase and anti-hyaluronidase activity of phosvitin.

2. Elastase from porcine pancreas and hyaluronidase from bovine testes were used to study the inhibitory activity of phosvitin. To elucidate the mechanism of enzyme inhibition, a Lineweaver-Burk plot was constructed.

3. Phosvitin inhibited elastase and hyaluronidase activity in a dose-dependent manner. The IC₅₀ value of phosvitin was 31.6 μg/ml and 1,270 μg/ml against elastase and hyaluronidase, respectively. The analysis of elastase and hyaluronidase kinetics indicated that the apparent Michaelis constant (_appK_m) was increased by phosvitin but the V_max value was not affected.

4. In conclusion, phosvitin exhibited competitive inhibitory activity against elastase and hyaluronidase. Thus, phosvitin could be used as a natural anti-aging agent in the cosmetics industry.     

Inhibition by melittin of phosphorylation by protein kinase C of annexin I from cow mammary gland

Protein kinase C (PKC) is a phosphotransferase activated by diacylglycerols, phospholipids and Ca(2+), that regulates a wide variety of biological functions by phosphorylating multiple protein substrates such as annexin I. Annexin I is a phospholipid/Ca (2+)-binding protein distributed in various tissues, including the mammary gland, and is thought to mediate the anti-inflammatory actions of glucocorticoids by inhibiting phospholipase A(2). Melittin, a phospholipase A(2) activator in bee venom, is known to inhibit PKC activity when lysine-rich histone is used as the substrate. The purpose of the present study was to examine whether phosphorylation by PKC of annexin I from cow mammary gland was inhibited by melittin. Melittin inhibited annexin I phosphorylation by PKC in a dose-dependent manner, and its IC(50) value (concentration causing 50% inhibition) was 0.8 microM. The phosphorylation of annexin I was also inhibited by the amphiphilic polypeptides mastoparan and polymyxin B, and their inhibitory effects were comparable to that of melittin. The surface-inactive polypeptide bacitracin was less effective. The inhibition by melittin was effectively reversed by the excess addition of phosphatidylserine, but not distinctly by 1-oleoyl-2-acetyl-sn-glycerol or Ca(2+), suggesting that melittin inhibited the phosphorylation of annexin I by interacting with phosphatidylserine. The inhibition by melittin of PKC phosphorylation of annexin I seems to be pathophysiologically important, because a melittin-like phospholipase A(2)-stimulatory protein is present in bovine endothelial cells.     

In vitro studies of the effect of propylthiouracil and ronnel on thyroxine-5'-monodeiodinase activity in steers

This study compares in vitro effects of propylthiouracil (PTU) and ronnel on the conversion of thyroxine (T4) to triiodothyronine (T3) in liver and kidney from Angus steers. Tissues were homogenized and incubated with T4 (1.3 microM) in the presence of 0 to 59 microM PTU or 0 to 49.7 microM ronnel. The T3 generated during a 30-min incubation was measured by radioimmunoassay. It was found that 1.47, 5.9 and 59 microM PTU decreased T4 to T3 conversion in liver and kidney by 62 and 88, 71 and 100, and 81 and 100%, respectively. The inhibition caused by 1.47 and 5.9 microM PTU was overcome by addition of 2 mM dithiothreitol (DTT). Ronnel in concentrations of 1.24, 6.22, 49.7 microM decreased T4 to T3 conversion in liver and kidney 46 and 45, 51 and 72, and 78 and 95%, respectively. However, with ronnel, the addition of DTT caused further inhibition. A Lineweaver-Burk plot of the data obtained using .32 to 6.43 microM T4 with 1.47 and 5.9 microM PTU or 6.22 and 12.44 microM ronnel indicated that PTU is an uncompetitive inhibitor (Ki = 1.67 microM) and ronnel is a noncompetitive (Ki = 15.5 microM) inhibitor of T4-5'-monodeiodination. The data suggest that decreased conversion of T4 to T3 by PTU or ronnel may be responsible for the increased plasma concentrations of T4 and slightly decreased plasma concentrations of T3 reported in steers treated with levels of both PTU and ronnel that are associated with growth stimulation.     

桑叶1-脱氧野尻霉素(DNJ)对α-蔗糖酶的抑制动力学研究

从桑叶中提取的1-脱氧野尻霉素(DNJ)作为糖苷酶抑制剂具有降血糖的功能。为了解明桑叶DNJ对α-蔗糖酶的作用机制,应用酶抑制动力学分析桑叶DNJ(纯度95%)对α-蔗糖酶的抑制特性。通过建立的α-蔗糖酶酶促反应体系表明,在α-蔗糖酶质量浓度≤500μg/mL、反应时间8min以内时,反应体系呈现良好的线性趋势;抑制活性检测表明,桑叶DNJ对α-蔗糖酶具有明显的抑制作用,其半抑制浓度(IC50)为5.67mg/mL;依据酶抑制动力学曲线特征推断桑叶DNJ对α-蔗糖酶具有明显可逆的非竞争性抑制作用,其抑制常数Ki为6.73mg/mL。     

Potency and selectivity of carprofen enantiomers for inhibition of bovine cyclooxygenase in whole blood assays

Whole blood in vitro assays were used to determine the potency and selectivity of carprofen enantiomers for inhibition of the isoforms of cyclooxygenase (COX), COX-1 and COX-2, in the calf. S(+)-carprofen possessed preferential activity for COX-2 inhibition but, because the slopes of inhibition curves differed, the COX-1:COX-2 inhibition ratio decreased from 9.04:1 for inhibitory concentration (IC)(10) to 1.84:1 for IC(95). R(-) carprofen inhibited COX-2 preferentially only for low inhibition of the COX isoforms (IC(10) COX-1:COX-2=6.63:1), whereas inhibition was preferential for COX-1 for a high level of inhibition (IC(95) COX-1:COX-2=0.20:1). S(+) carprofen was the more potent inhibitor of COX isoforms; potency ratios S(+):R(-) carprofen were 11.6:1 for IC(10) and 218:1 for IC(90). Based on serum concentrations of carprofen enantiomers obtained after administration of a therapeutic dose of 1.4mg/kg to calves subcutaneously, S(+)-carprofen concentrations exceeded the in vitro IC(80) COX-2 value for 32h and the IC(20) for COX-1 for 33h. The findings are discussed in relation to efficacy and safety of carprofen in calves.     

Biological activity of dihydroartemisinin in canine osteosarcoma cell lines

OBJECTIVE: To evaluate the biological activity of dihydroartemisinin on canine osteosarcoma cell lines in vitro. SAMPLE POPULATION: 4 canine osteosarcoma cell lines. PROCEDURES: Cell viability assays were performed on canine osteosarcoma cell lines OSCA2, OSCA16, OSCA50, and D17 after 24, 48, and 72 hours of treatment with dihydroartemisinin at concentrations of 0.1 to 100 microM. Apoptosis was assessed by use of an ELISA for free nuclosomal DNA fragmentation and by western blot analysis for cleavage of caspase 3. Cell cycle analysis was performed by use of staining with propidium iodide and flow cytometry. Detection of reactive oxygen species (ROS) was conducted in the D17 cell line by use of 6-carboxy-2',7'-dihydrofluorescein diacetate and flow cytometry. RESULTS: The concentration of dihydroartemisinin required for 50% inhibition of cell viability (IC50) was achieved in all 4 canine osteosarcoma cell lines and ranged from 8.7 to 43.6 microM. Induction of apoptosis was evident as an increase in nucleosomal DNA fragmentation, cleavage of caspase 3, and an increase in the population in the sub G0/G1 phase of the cell cycle detected by flow cytometry. Exposure to dihydroartemisinin also resulted in a decrease in the G0/G1 population. Iron-dependent generation of ROS was detected in dihydroartemisinin-treated D17 cells; ROS generation increased in a dose-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Incubation with dihydroartemisinin resulted in biological activity against canine osteosarcoma cell lines, which included induction of apoptosis and arrest of the cell cycle. Clinical trials of dihydroartemisinin in dogs with osteosarcoma should be conducted.     

桑枝皮多糖的提取及组成成分与体外生物活性分析

为了更好地发掘桑枝皮的药用价值,采用热水浸提法从桑枝皮中分离提取多糖,其得率达到28.5%。采用薄层层析(TLC)法和非衍生化高效液相色谱(HPLC)法分析桑枝皮多糖的组成成分,初步推定桑枝皮多糖为一种杂多糖,由鼠李糖、木糖、阿拉伯糖、甘露糖、葡萄糖和半乳糖等单糖组成。桑枝皮多糖的体外生物活性试验显示其具有较强的对DPPH自由基的清除能力(IC50为1.7mg/mL)和对α-葡萄糖苷酶的抑制活性(IC50为0.298mg/mL)。上述结果提示桑枝皮多糖是降血糖和抗氧化的天然药物资源。     

Evaluation of the effects of histone deacetylase inhibitors on cells from canine cancer cell lines

OBJECTIVE: To determine whether exposure of canine cancer cells to histone deacetylase (HDAC) inhibitors S(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)benzamide (OSU-HDAC42) or suberoylanilide hydroxamic acid (SAHA) results in increased histone acetylation and decreased cell viability and whether any changes in viability involve induction of apoptosis or alterations in progression of the cell cycle. Sample POPULATION: 9 canine cancer cell lines. PROCEDURES: Cells from 9 canine cancer cell lines were treated with dimethyl sulfoxide vehicle, OSU-HDAC42, or SAHA, then assays of cell viability were performed. Histone acetylation was assessed by use of western blot analysis. Apoptosis was assessed via ELISA to detect fragmentation of cytoplasmic nucleosomal DNA and western blot analysis to detect cleavage of caspase 3. Cell cycle analysis was performed by use of propidium iodide staining and flow cytometry. RESULTS-Concentrations of OSU-HDAC42 and SAHA required to achieve 50% inhibition of cell viability (IC(50)) were reached in cells of 6 and 4 canine cancer cell lines, respectively, and ranged from approximately 0.4 to 1.3 microM for OSU-HDAC42 and 0.6 to 4.8 microM for SAHA. Cells from T-cell lymphoma, mast cell tumor, osteosarcoma, and histiocytic sarcoma lines were most sensitive to HDAC inhibition, with IC(50)s of < 1 microM for OSU-HDAC42 and < 5 microM for SAHA. Induction of apoptosis was indicated via cleavage of caspase 3 and increases in cytoplasmic nucleosomes and the subG(1) cell population. CONCLUSIONS AND CLINICAL RELEVANCE: Micromolar concentrations of HDAC inhibitors OSU-HDAC42 and SAHA induced histone acetylation, cytotoxicity, and apoptosis in canine cancer cells. In general, OSU-HDAC42 was more potent than SAHA.     

In vitro efficacy of ganciclovir, cidofovir, penciclovir, foscarnet, idoxuridine, and acyclovir against feline herpesvirus type-1

OBJECTIVE: To establish the in vitro efficacy of 4 novel drugs (ie, ganciclovir, cidofovir, penciclovir, and foscarnet) against feline herpesvirus type-1 (FHV-1) and compare their antiviral efficacy with that of acyclovir and idoxuridine. SAMPLE POPULATION: Cultured Crandell-Reese feline kidney (CRFK) cells and FHV-1 strain 727 PROCEDURE: For each drug, antiviral effect was estimated by use of conventional plaque-reduction assays, and inhibitory concentration 50 (IC50; drug concentration at which plaque numbers were reduced by 50% relative to the number of plaques for nontreated control wells) was calculated. To determine whether observed antiviral effects were related to alterations in the number or viability of CRFK cells, cytotoxicity assays were performed at 1, 2, and 10 times the median IC50 for each antiviral drug. RESULTS: Median IC50 for each drug was as follows: ganciclovir, 5.2 microM; cidofovir, 11.0 microM; penciclovir, 13.9 microM; foscarnet, 232.9 microM; idoxuridine, 4.3 microM; and acyclovir, 57.9 microM. Obvious changes in morphologic characteristics, confluence, or viability of CRFK cells were not observed at concentrations up to and including 2 times the IC50 for each drug. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro efficacy of idoxuridine and ganciclovir against FHV-1 was approximately equivalent and about twice that of cidofovir and penciclovir. Foscarnet appeared to be comparatively ineffective. Given the reasonable clinical efficacy of idoxuridine in cats infected with FHV-1, clinical trials of ganciclovir, cidofovir, and penciclovir or their prodrug forms appear to be warranted.     

Maedi-visna virus, a model for in vitro testing of potential anti-HIV drugs

A series of beta-D- and beta-L-cytidine analogues were evaluated for their inhibitory effect on the replication of maedi-visna virus (MVV) strains KV1772 and MV1514 cultured on sheep choroid plexus cells and the sheep chondrocyte cell line G81092, respectively. Eleven cytidine analogues were selected for the anti-viral test. Five of them belong to the family of the 2',3'-dideoxycytidine analogues, well known for their activity against human immunodeficiency virus (HIV). The others, all newly synthesized, were potential anti-viral and/or anti-leukemic agents. None of the compounds under study had a toxic effect in both anti-viral assay systems up to a 300 microM concentration. Based on the cytopathic effects (CPE), the virus replication was completely inhibited by the five 2',3'-dideoxycytidine analogues at a concentration of 50 microM, whereas the others six newly synthesized compounds induced titre reductions of 4-5 log units. The effective concentration causing 50% reduction of CPE (EC50) was of 5 microM for the five 2',3'-dideooxycytidine analogues and for beta-L-XyloFc, whereas the value of 50 microM was found for the b-L-XyloC and the four 5-azacytidine compounds tested. All these data reveal a good correlation between inhibition of MVV replication by several nucleoside cytidine analogues and their reported anti-HIV activity.     

Benzodiazepines inhibit the acetylcholine receptor-operated potassium current (IK.ACh) by different mechanisms in guinea-pig atrial myocytes

The anticholinergic effects of 7 benzodiazepines, bromazepam, camazepam, chlordiazepoxide, diazepam, lorazepam, medazepam and triazolam, were compared by examining their inhibitory effects on the acetylcholine receptor-operated potassium current (I(K).(ACh)) in guinea-pig atrial myocytes. All of these benzodiazepines (0.3-300 μM) inhibited carbachol (1 μM)-induced I(K).(ACh) in a concentration-dependent manner. The ascending order of IC(50) values for carbachol-induced I(K).(ACh) was as follows; medazepam, diazepam, camazepam, triazolam, bromazepam, lorazepam and chlordiazepoxide (>300 μM). The compounds, except for bromazepam, also inhibited I(K).(ACh) activated by an intracellular loading of 100 μM guanosine 5'-[γ-thio]triphosphate (GTPγS) in a concentration-dependent manner. The ascending order of IC(50) values for GTPγS-activated I(K).(ACh) was as follows; medazepam, diazepam, camazepam, lorazepam, triazolam chlordiazepoxide (>300 μM) and bromazepam (>300 μM). To clarify the molecular mechanism of the inhibition, IC(50) ratio, the ratio of IC(50) for GTPγS-activated I(K).(ACh) to carbachol-induced I(K).(ACh), was calculated. The IC(50) ratio for camazepam, diazepam, lorazepam, medazepam and triazolam was close to unity, while it for chlordiazepoxide could not be calculated. These compounds would act on the GTP binding protein and/or potassium channel to achieve the anticholinergic effects in atrial myocytes. In contrast, since the IC(50) ratio for bromazepam is presumably much higher than unity judging from the IC(50) values (104.0 ± 30.0 μM for carbachol-induced I(K).(ACh) and >300 μM for GTPγS-activated I(K).(ACh), it would act on the muscarinic receptor. In summary, benzodiazepines had the anticholinergic effects on atrial myocytes through inhibiting I(K).(ACh) by different molecular mechanisms.     

The loop diuretic bumetanide as a tool in physiology and pharmacology]

Loop diuretics are derivatives of 4-sulfamoylbenzoic acid, which derived originally from sulfonamides. Their diuretic effect is due to the inhibition of the Na-K-Cl-cotransport system in the distal part of Henle's loop. The compounds react with different affinity with the chloride binding site of the transporter. Bumetanide is among the most potent blockers with an IC50 of 0.2 microM. The compound was developed by P. W. FEIT, 1971; the saluretic response was described first by H. H. FREY 1972. Bumetanide has outgrown to become a tool for physiologists and pharmacologists in renal transport research. The compound has essentially contributed to the elucidation of the mechanisms of volume regulation of cells. Bumetanide is taken up into tubule cells and hepatocytes by active transport. The uptake in tubule cells ist mediated by an organic anion transporter, which is involved in the renal secretion of drugs. In the liver bumetanide is transporter by the bile acid carrier. The carrier is a multispecific drug transporter, too. It is not yet known, whether both drug transport systems contain identical membrane proteins. For this purpose bumetanide is currently used to investigate by photoaffinity labeling and functional expression cloning molecular principles of the drug elimination in kidney and liver.     

The in vitro secretion of acetylcholinesterase by adult stages of Heligmosomoides polygyrus: the effects of broad-spectrum anthelmintics

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 +/- 0.07 mumol min-1 l-1 mg-1) than for female (0.56 +/- 0.04 mumol min-1 mg-1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug-free medium (RPMI medium supplemented with 0.5% BSA) for 24 h or continuously exposed to the drugs in supplement-free medium (24 h), the concentration- and time-dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50% (IC50) relative to drug-free controls were estimated. The IC50 values ranged from 0.012 microM (IVM) to 2.96 microM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad-spectrum anti-nematodal activity is discussed.     

Prognostic evaluation of Ki67 threshold value in canine oral melanoma

Oral melanoma is a common canine cancer with a historically poor prognosis. Recent evidence suggests that a subset of cases may have a more favorable outcome, defined as long-term survival in the absence of intervention other than initial surgery. Traditional histological parameters have had prognostic significance in some studies but not in others, potentially due to interobserver variation. We evaluated the prognostic utility of Ki67 immunohistochemistry in a group of 79 canine oral melanomas using a technique easily applied in a veterinary diagnostic laboratory. A threshold Ki67 value of >19.5 had a sensitivity and specificity of 87.1% and 85.4%, respectively, at predicting death or euthanasia due to melanoma by 1 year postdiagnosis. Threshold values for classical histological parameters were also identified for most cases and were >4 (>30%; sensitivity = 83.9%, specificity = 86.0%) for the nuclear atypia score and >4/10 hpfs (sensitivity = 90.3%, specificity = 84.4%) for the mitotic index. In this study, the percentages correctly classified with respect to death by 1 year postdiagnosis were comparable for Ki67 (86.1%, 68/79), the nuclear atypia score (86.3%, 63/73), and the mitotic index (86.8%, 66/76). High pigmentation (>50%) had a high negative predictive value of 90.9% (18/20), but overall, only 61.0% (47/77) of cases could be correctly classified by this parameter. Based on these results, we recommend a panel of prognostic parameters, including the nuclear atypia score, the mitotic index, Ki67, and pigmentation quantification to more accurately predict the likely outcome of canine oral melanomas.     

Antioxidative and antigenotoxic effects of Japanese horse chestnut (Aesculus turbinata) seeds

Japanese horse chestnut seed extract (HCSE) dose-dependently inhibited the autooxidation of linoleic acid (IC(50): 0.2 mg/ml), and the inhibition was almost complete at a concentration of 1 mg/ml. The HCSE scavenged DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals and superoxide anions with EC(50)s of 0.65 and 0.21 mg/ml, respectively. However, it had no effect on hydrogen peroxide. The HCSE inhibited the genotoxicities of furylfuramide, N-methyl-N-nitrosourea, methyl methanesulfonate, mitomycin C, 2-aminoanthracene and aflatoxin B1 at a concentration of 1 mg/ml or more. Total polyphenol content of the HCSE was 21 mg/g (13 mg/g-seeds). These results indicate that the Japanese horse chestnut seed is an antioxidative and antimutagenic botanical resource.     

Laidlomycin butyrate--an ionophore with enhanced intraruminal activity

The polyether ionophore, laidlomycin, and several acyl derivatives were tested for their ability to favorably alter fermentation in two types of in vitro rumen fluid incubations. Dose response data were used to estimate the concentration (microgram/ml) of each ionophore required for either 50% maximal enhancement of propionic acid production (EC50) or 50% maximal inhibition of lactic acid production (IC50). Acylation of laidlomycin with straight-chain acyl groups from two to 12 carbon atoms tended to improve the potency of laidlomycin, especially for inhibiting lactic acid production. Comparative incubations using laidlomycin butyrate, laidlomycin and monensin indicated that both laidlomycin butyrate (EC50 = .3) and monensin (EC50 = .7) were more potent enhancers of propionic acid production than laidlomycin (EC50 = 2.0; P less than .05). Laidlomycin butyrate (IC50 = .3) was a more potent inhibitor of lactic acid production than either laidlomycin (IC50 = 1.8) or monensin (IC50 = 1.3; P less than .05). In a continuous culture experiment, three chemostats each received laidlomycin butyrate or monensin at the rate of .5 micrograms/ml of effluent/d while two flasks remained as control. Propionic acid production was increased (P less than .01) from 22.9 mmol/d in control flasks to 30.5 and 33.7 mmol/d in flasks treated with monensin and laidlomycin butyrate, respectively. Concomitant decreases in production rates of acetic, butyric and isovaleric acids also were observed (P less than .01). Thirty-six steers were used in a 56-d trial to evaluate effects of laidlomycin butyrate and monensin, at 33 mg/kg of diet, on feedlot performance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of the beta-adrenergic receptor agonist zilpaterol on the LPS-induced production of TNF-alpha in vitro and in vivo

In this study the anti-inflammatory properties of zilpaterol, a beta2-adrenergic receptor (AR) agonist specifically developed as a growth promoter in cattle were investigated. Although zilpaterol has a different structure compared with the beta2-AR agonists known to date, it was noted that it was able to bind to both the beta2-AR (Ki = 1.1 x 10(-6)) and the beta1-AR (Ki = 1.0 x 10(-5)). Using lipopolysaccharide (LPS)-exposed U937 macrophages, the production of cyclic adenosine-3',5'-cyclic monophosphate (cAMP) and tumor necrosis factor alpha (TNF-alpha) were investigated. Zilpaterol inhibited TNF-alpha release and induced intracellular cAMP levels in a dose-dependent manner. The inhibition of TNF-alpha release and induction of cAMP production was mainly mediated via the beta2-AR, as indicated by addition of beta1- and beta2-specific antagonists. The effects of zilpaterol were investigated in LPS-treated male Wistar rats after pretreatment with zilpaterol. Zilpaterol dosed at 500 microg/kg body weight reduced the TNF-alpha plasma levels. In conclusion, zilpaterol is a beta2-adrenergic agonist and an inhibitor of TNF-alpha production induced by LPS both in vivo and in vitro.     

Pharmacological characterization of alpha1-adrenoceptor subtypes in the bovine tail artery

Ioudina, M. V., Dyer, D. C. Pharmacological characterization of alpha1-adrenoceptor subtypes in the bovine tail artery. J. vet. Pharmacol. Therap. 25, 363-369. The purpose of this study was to identify the alpha1-adrenoreceptor subtypes present in the bovine tail artery which mediate contractions to adrenergic agonists. A61603, an alpha1A-selective agonist, was more potent compared with norepinephrine and phenylephrine. The pKA value of A61603 was 6.93 +/- 0.19 microM (n=6). Antagonists, BMY 7378, WB 4101 and 5-methylurapidil, caused a parallel shift to the right of the concentration-response curve to A61603 with pA2 values of 6.62, 9.27 and 8.86, respectively. Prazosin, BMY 7378 and WB 4101 inhibited phenylephrine induced contraction with pA2 values of 9.47, 7.17 and 9.73, respectively. The pA2 values obtained for 5-methylurapidil, WB 4101, BMY 7378 and prazosin against alpha1-adrenoceptor agonists were significantly correlated with pKi values (Zhu, Zhang & Han, 1997, Eur. J. Pharmacol.329, 55-61) for the cloned alpha1a-adrenoceptor but not with the cloned alpha1b- or alpha1d-adrenoceptor. BMY 7378, a selective alpha1D-adrenoceptor antagonist, was significantly more potent against the nonsubtype selective agonist phenylephrine than to A61603. Chloroethylclonidine (50 microM for 10 min) did not affect contractile responses to A61603, but caused a significant inhibition of contractile responses to phenylephrine. In conclusion, it appears that alpha1A- and alpha1D-adrenoceptors play a role in adrenergic mediated contraction in the bovine tail artery.     

Influence of marbofloxacin on the pharmacokinetics and pharmacodynamics of tolfenamic acid in calves

Pharmacokinetic and pharmacodynamic properties of tolfenamic acid (TA) in calves were determined in serum and fluids of inflamed (carrageenan administered) and non-inflamed subcutaneously implanted tissue cages after intramuscular administration both alone and in combination with marbofloxacin (MB). MB significantly altered the pharmacokinetics of TA: mean values were Cmax = 2.14 and 1.64 microg/mL, AUC = 27.38 and 16.80 microg.h/mL, Vd(area)/F = 0.87 and 1.17 L/kg, and ClB/F = 0.074 and 0.128 L/kg/h, respectively, after administration of TA alone and TA + MB. T(1/2)K10 and MRT were not significantly different for the two treatments. The pharmacodynamic properties of TA were not influenced by MB co-administration, in spite of the alterations in some TA pharmacokinetic parameters. TA inhibited prostaglandin E2 (PGE2) synthesis in vivo in inflammatory exudate by 50-88% for up to 48 h after both TA treatments. Inhibition of synthesis of serum thromboxane B2 (TxB2) ex vivo ranged from 40 to 85% up to 24 h after both TA and TA + MB. From the derived pharmacokinetic and eicosanoid inhibition data for TA, pharmacodynamic parameters for serum TxB2 and exudate PGE2 inhibition expressing efficacy (Emax = 78.1 and 97.5%), potency (IC50 = 0.256 and 0.265 microg/mL), sensitivity (N = 1.96 and 2.29) and the pharmacokinetic parameter equilibration time (t(1/2)K(e0) = 0.695 and 24.0 h), respectively, were determined. In this model TA was a nonselective inhibitor of cyclo-oxygenase (COX) (COX-1:COX-2 IC50 ratio = 1.37). TA, both alone and co-administered with MB, did not affect leucocyte numbers in exudate, transudate or blood. Partial attenuation of skin temperature rise over inflamed tissue cages and reduction of zymosan-induced skin swelling were recorded after administration of TA and TA + MB with no significant differences between the two treatments. These data provide a basis for the rational use of TA in combination with MB in calf medicine.