Development of a high-throughput fluorescence polarization assay for the discovery of EZH2-EED interaction inhibitors.

Aberrant activity of enhancer of zeste homolog 2 (EZH2) is associated with a wide range of human cancers. The interaction of EZH2 with embryonic ectoderm development (EED) is required for EZH2's catalytic activity. Inhibition of the EZH2-EED complex thus represents a novel strategy for interfering with the oncogenic potentials of EZH2 by targeting both its catalytic and non-catalytic functions. To date, there have been no reported high-throughput screening (HTS) assays for inhibitors acting at the EZH2-EED interface. In this study, we developed a fluorescence polarization (FP)-based HTS system for the discovery of EZH2-EED interaction inhibitors. The tracer peptide sequences, positions of fluorescein labeling, and a variety of physicochemical conditions were optimized. The high Z' factors (>0.9) at a variety of DMSO concentrations suggested that this system is robust and suitable for HTS. The minimal sequence requirement for the EZH2-EED interaction was determined by using this system. A pilot screening of an in-house compound library containing 1600 FDA-approved drugs identified four compounds (apomorphine hydrochloride, oxyphenbutazone, nifedipine and ergonovine maleate) as potential EZH2-EED interaction inhibitors.

Oxyphenbutazone promotes cytotoxicity in rats and Hep3B cellsvia suppression of PGE2 and deactivation of Wnt/ß-catenin signaling pathway.

Hepatocellular carcinoma (HCC) is the fifth leading cause of death and is generally typified by elevated liver enzyme biomarkers, antioxidants, and chronic inflammation of hepatocytes. Although currently available drugs have shown remarkable alleviation of the cancerous condition, but at the same time they present a more severe challenge of toxic effects due to chemotherapy. Therefore, in order to bring more patient-compliant therapy, we aimed to refurbish the use of a COX inhibitor, oxyphenbutazone (OPB), with low dose of methotrexate (MTX) to treat diethyl nitrosamine (DENA)-induced HCC in Wistar rats and in Hep3B cells. Hep3B cells were subjected to assays like in vitro cytotoxicity, DNA synthesis, and caspase activity. The combination index was also evaluated, succeeding the cytotoxicity assay, to analyze the possible synergism. For in vivo study, Wistar strain male rats were given single intraperitoneal dose of DENA (200 mg/kg) and were supplied with sodium phenobarbital (0.1% in tap water) for promoting tumorigenesis throughout the study. MTX (2.5 and 5.0 mg/kg/week, ip) and OPB (70 mg/kg/week, po in two divided doses) were administered to the treatment groups from 3rd week till the termination of study. Several biochemical parameters including biomarkers of liver function, antioxidant enzymes, and histopathological examination of liver cells were tested. Significant synergism was witnessed in the cytotoxicity assay when Hep3B cells received varied dose combination treatment of MTX (0.25, 0.5, or 1.0 µmol/L) and OPB (2.5, 5.0, or 7.5 µmol/L). MTX (0.5 and 1.0 µmol/L) in combination with OPB (5.0 or 7.5 µmol/L) inhibited the cell proliferation as BrdU incorporation was quite low in DNA synthesis analysis, as well as caspase-9/-3 cascade was activated which led to apoptosis of cancer cells. Co-treatment with MTX and OPB exerted potential anticancer activity in rats than either of the drugs alone. Administration of combination therapy harmonized the DENA-induced elevation of serum biochemical parameters, including but not limited to, α-fetoprotein (AFP), alanine- and aspartate-aminotransferase, alkaline phosphatase, vascular endothelial growth factor (VEGF), and antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and lipid per oxidation (LPO). All these results were optimally substantiated by histopathological examination. As evident COX-2 catalyzes the synthesis of PGE2, needed in the activation of Wnt/ß-catenin pathway, which in turn is responsible for activating the transcriptional proteins required for higher degree of cell division and thence growth. Therefore, inhibition of COX-2 by our novel combination infers that even low doses of MTX can elucidate noticeable anticancer activity when paired with OPB.

Nonsteroidal anti-inflammatory drug sensitizes Mycobacterium tuberculosis to endogenous and exogenous antimicrobials.

Existing drugs are slow to eradicate Mycobacterium tuberculosis (Mtb) in patients and have failed to control tuberculosis globally. One reason may be that host conditions impair Mtb's replication, reducing its sensitivity to most antiinfectives. We devised a high-throughput screen for compounds that kill Mtb when its replication has been halted by reactive nitrogen intermediates (RNIs), acid, hypoxia, and a fatty acid carbon source. At concentrations routinely achieved in human blood, oxyphenbutazone (OPB), an inexpensive anti-inflammatory drug, was selectively mycobactericidal to nonreplicating (NR) Mtb. Its cidal activity depended on mild acid and was augmented by RNIs and fatty acid. Acid and RNIs fostered OPB's 4-hydroxylation. The resultant 4-butyl-4-hydroxy-1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (4-OH-OPB) killed both replicating and NR Mtb, including Mtb resistant to standard drugs. 4-OH-OPB depleted flavins and formed covalent adducts with N-acetyl-cysteine and mycothiol. 4-OH-OPB killed Mtb synergistically with oxidants and several antituberculosis drugs. Thus, conditions that block Mtb's replication modify OPB and enhance its cidal action. Modified OPB kills both replicating and NR Mtb and sensitizes both to host-derived and medicinal antimycobacterial agents.

Chemopreventive activities of etodolac and oxyphenbutazone against mouse skin carcinogenesis.

Previous cancer chemoprevention studies have demonstrated that the non-steroidal anti-inflammatory drugs (NSAIDs) can be effective in suppressing the development of various human malignancies. Recently we identified the possible anti-tumor promoting potentials of 14 new NSAIDs in the Epstein-Barr virus early antigen activation assay induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study we report the inhibition of 7,12-dimethylbenz (a) anthracene (DMBA) induced two-stage mouse skin carcinogenesis by etodolac (ETD), one of the most potent NSAIDs identified in our in vitro cancer chemopreventive screening of this group of drugs. Topical administration of ETD at a very low dose of 85 nmol showed a significant decrease in both tumor incidence and burden. This effect is also accompanied by a delay in the tumor latency period. Since ETD showed potent chemopreventive activity in both in vitro and in vivo studies, it warrants prompt consideration for trial in humans as a potential cancer chemopreventive agent. We also investigated oxyphenbutazone (OPB) another commonly used NSAID for its cancer chemopreventive effect on peroxynitrite (PN) induced-TPA promoted skin tumors in the mouse. Following tumor initiation with 390 nmol of PN, the skin tumor promotion with 1.7 nmol of TPA was significantly inhibited by oral administration of 0.0025% OPB. The results demonstrate that OPB is a potent cancer chemopreventive agent in the highly sensitive in vivo mouse test model we used.

4-Hydroxy-oxyphenbutazone is a potent inhibitor of cytokine production.

4-Hydroxy-oxyphenbutazone (4OH-OPB), is currently in phase II trials for its immunosuppressive effect in patients with rheumatoid arthritis. 4OH-OPB and other compounds related to phenylbutazone were tested for their effect on in vitro cytokine production by monocytes and lymphocytes present in peripheral mononuclear cells (PBMC) or whole blood (WB) cultures, and compared against phenylbutazone and oxyphenbutazone, two known anti-inflammatory drugs. In PBMC cultures, 4OH-OPB was by far the most potent inhibitor, and both monokines and Th1 and Th2 lymphokines were efficiently inhibited at low concentrations. In WB cultures, 4OH-OPB was less effective than in PBMC cultures, but was still the best inhibitor of lymphokine production and, furthermore, was the only inhibitor of monokine production. The increase in 4OH-OPB concentration needed to induce the same inhibition of cytokine production in WB as in PBMC culture could be mimicked by the addition of erythrocytes to the PBMC cultures. Experiments with radioactively-labeled 4OH-OPB suggest that 4OH-OPB is taken up very rapidly into erythrocytes and is secreted by the erythrocytes with much slower kinetics via a multidrug-resistance-associated protein. The secreted compound is most likely structurally different from 4OH-OPB, as in PBMC and WB cultures, the inhibition of cytokine production seems to be caused by a different mechanism. In PBMC cultures, the inhibition of cytokine production is accompanied by a loss of cell viability, while this is not the case when 4OH-OPB inhibits cytokine production in WB. Our data suggest that 4OH-OPB may be useful as an immunosuppressive drug for patients with inflammatory diseases.

Pretreatment of astrocytes with interferon-alpha/beta impairs interferon-gamma induction of nitric oxide synthase.

Excessive nitric oxide/peroxynitrite generation has been implicated in the pathogenesis of multiple sclerosis, and the demonstration of increased astrocytic nitric oxide synthase activity in the postmortem brain of multiple sclerosis patients supports this hypothesis. Exposure of astrocytes, in primary culture, to interferon-gamma results in stimulation of nitric oxide synthase activity and increased nitric oxide release. In contrast to interferon-gamma, interferon-alpha/beta had a minimal effect on astrocytic nitric oxide formation. Furthermore, pretreatment of astrocytes with interferon-alpha/beta inhibited (approximately 65%) stimulation by interferon-gamma of nitric oxide synthase activity and nitric oxide release. Treatment with interferon-alpha/beta at a concentration as low as 10 U/ml caused inhibition of mitochondrial cytochrome c oxidase. Furthermore, the damage to cytochrome c oxidase was prevented by the putative interferon-alpha/beta receptor antagonist oxyphenylbutazone. In view of these observations, our current hypothesis is that the mitochondrial damage caused by exposure to interferon-alpha/beta may impair the ability of astrocytes to induce nitric oxide synthase activity on subsequent interferon-gamma exposure. These results may have implications for our understanding of the mechanisms responsible for the therapeutic effects of interferon-alpha/beta preparations in multiple sclerosis.

Non-phenolic linear diarylheptanoids from Curcuma xanthorrhiza: a novel type of topical anti-inflammatory agents: structure-activity relationship.

The topical anti-inflammatory activity of three non-phenolic linear 1,7-diarylheptanoids, previously isolated from a Thai medicinal plant, Curcuma xanthorrhiza (Zingiberaceae) and four new semi-synthetic derivatives of the naturally occurring compounds were assessed in the murine model of ethyl phenylpropiolate-induced ear edema. The naturally occurring compound 1E,3E,1,7-diphenylheptadien-5-one (6) exerted the most potent anti-inflammatory activity, with an ID50 value of similar magnitude to that of the reference drug oxyphenbutazone (67 vs. 46 micrograms/ear, respectively). None of the semi-synthetic diarylheptanoids was more active than 6. The chemical structures and pharmacological data of the natural and semi-synthetic derivatives identified a distinct structure-activity relationship. The degree of unsaturation in positions 1 and 3, and the nature of the oxygenated functional group in position 5 of the C7-chain were found to play significant roles in determining the observed in vivo activity. Based on these findings, the non-phenolic linear 1,7-diarylheptanoids-are proposed to represent a novel class of topical anti-inflammatory agents.

Decrease of inorganic blood sulfate following treatment with selected antirheumatic drugs: potential consequence for articular cartilage.

The elimination kinetics of inorganic blood sulfate in mice was followed for four hours after a single, oral administration of an antirheumatic drug. Sodium salicylate, aspirin, diflunisal and benorylate, all in a dose of 1.25 mmol/kg, reduced the sulfate level to the less than half that of control. This phenomenon was also demonstrated by phenylbutazone, oxyphenbutazone (both 1 mmol/kg), chloroquine diphosphate (0.6 mmol/kg) and tiaprofenic acid (0.02-0.35 mmol/kg). Niflumic acid (1.08 mmol/kg), piroxicam (0.03 mmol/kg), indomethacin (6.10(-3) mmol/kg), diclofenac (5.10(-3) mmol/kg), ketoprofen (0.2 mmol/kg), naproxen (0.08 mmol/kg) and ibuprofen (0.24 mmol/kg) possessed no sulfate lowering properties. The potential relevance of the use of sulfate lowering drugs for articular cartilage integrity is discussed in the light of what is already known about this subject.

Anti-inflammatory activity of Teucrium polium.

Teucrium polium is widely used by the folk-medicine practitioners in Saudi Arabia for the treatment of inflammations, rheumatism, diabetes and ulcers. Scientific reports are lacking to confirm these activities. The present study reports the effect of ethanolic extract of T. polium on carrageenan-induced acute inflammation, cotton-pellet granuloma and some of the biochemical parameters. The ethanolic extract of Teucrium polium at a dose of 500 mg/kg body weight produced significant inhibition of carrageenan-induced inflammation and cotton-pellet granuloma. Biochemical studies showed a significant decrease in glucose level. The presence of flavonoids and sterols might be responsible for the anti-inflammatory activity of this plant. Further studies on the fractionation of the phytoconstituents, and on their mechanism of action are in progress.

Efeito de antiinflamatórios sobre o conteúdo histamínico pulmonar de ratos submetidos ao edema pulmonar agudo adrenalínico / Effect of antiinflamatory drugs on histamine pulmonary contents of rats submitted to acute adrenaline pulmonary edema

O conteúdo histamínico pulmonar de ratos submetidos ao edema pulmonar agudo adrenalínico näo foi afetado pelos antiinflamatórios tremaril e ácido acetilsalicílico. Porém, os animais previamente tratados com indometacina, na dose única de 50 mg/kg, assim como nas três doses de 25 mg/kg, apresentaram uma variaçäo estatisticamente significativa do conteúdo histamínico pulmonar. O mesmo fato foi observado com os animais pré-tratados com fenazona (10, 40 e 80 mg/kg). Os animais pré-tratados com fenilbutazona, nas doses de 10 e 80 mg/kg e aqueles com oxifenilbutazona (20 mg/kg) apresentaram uma variaçäo estatisticamente significativa do conteúdo histamínico pulmonar

Actions of non-steroidal anti-inflammatory drugs on equine leucocyte movement in vitro.

The direct effects of four non-steroidal anti-inflammatory drugs (NSAIDs) on equine polymorphonuclear (PMN) and mononuclear (MN) leucocyte movement were investigated using two in vitro assay systems. The Boyden chamber microfilter technique measures both chemokinetic and chemotactic locomotion, and the agarose microdroplet assay measures solely chemokinesis. Zymosan-activated plasma (ZAP) and the synthetic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) were used as standard chemoattractants for PMN and MN leucocytes, respectively. The actions of six concentrations of each NSAID, indomethacin (50 microM-10 mM), phenylbutazone (10 microM-1 mM), oxyphenbutazone (2.5 microM-500 microM) and flunixin (0.1 microM-50 microM), in suppressing cell movement induced by ZAP and FMLP were investigated. All four drugs exerted inhibitory effects on induced movement of both cell types in the Boyden chamber assay, usually in a concentration-dependent manner, although oxyphenbutazone action on PMN cells occurred only at the highest concentration tested. Significant inhibition of PMN and MN cell locomotion was produced by indomethacin, flunixin and oxyphenbutazone, and inhibition of PMN movement by phenylbutazone occurred in the agarose microdroplet assay. Flunixin was the most potent of the four drugs investigated in both assay systems. The findings may be of importance to the use of phenylbutazone and flunixin as NSAIDs in equine medicine, since the concentrations used were similar to concentrations of both drugs and the phenylbutazone metabolite oxyphenbutazone previously reported to occur in equine plasma and inflammatory exudate.

Evidence for a role of cyclooxygenase (prostaglandin synthetase) and prostaglandins in the sperm acrosome reaction and fertilization.

Three cyclooxygenase (prostaglandin synthetase) inhibitors, indomethacin, phenylbutazone, and oxyphenbutazone, decreased fertilization in vitro when mixed with capacitated mouse spermatozoa before addition of the treated gametes to oocytes. Fertilization was inhibited whether the oocytes were intact, follicle cell-free, or both follicle cell-free and zona-free. At various concentrations of inhibitor, no effect was observed on the motility or forward progression of the spermatozoa. These cyclooxygenase inhibitors also decreased the guinea pig acrosome reaction. Inhibition of the acrosome reaction did not occur when a mixture of the prostaglandins (PGE2 or PGF2 alpha) and one of the inhibitors was added to the spermatozoa. Alone, these prostaglandins tended to enhance the rate at which the acrosome reaction took place. Lowered calcium levels reduced the occurrence of the acrosome reaction, an effect that could be reversed at least partially by the addition of PGE2. Even in the nominal absence of calcium, some acrosome reaction took place when PGE2 was present in the medium. These results support an essential role for cyclooxygenase and arachidonic acid metabolites, including prostaglandins, in the events leading to the acrosome reaction and fertilization.

Reactions of oxyphenbutazone with active oxygen species.

The ability of Oxyphenbutazone (a non-steroidal antiinflammatory drug) to react with singlet oxygen and superoxide anions, possible mediators of the damage to the lipids of the cell membranes during inflammation was studied. Oxyphenbutazone inhibited the reduction of nitroblue tetrazolium in aerobic riboflavin-photosensitized oxidation of methionine, but did not influence the cytochrome C-reduction by superoxide-generating system xanthine-xanthine oxidase. Oxyphenbutazone was photooxidized in the presence of Rose Bengal, the latter being a photosensitizer. The increase of the reaction rate of Oxyphenbutazone-oxidation in D2O as compared to H2O, as well as the inhibition of oxidation by singlet oxygen-quencher sodium azide confirmed the participation of singlet oxygen in this process. It was found that Oxyphenbutazone reacted with singlet oxygen, but did not react with superoxide anions. This was supported by the observed protection of erythrocyte membranes from the hemolytic action of the singlet oxygen-generating system Rose Bengal + light.

Effects of phenylbutazone and oxyphenbutazone on acidic drug detection in high performance thin layer chromatographic systems.

Interference or "masking" in thin layer chromatography occurs when the presence of one drug on a thin layer plate physically obscures or interferes with the detection of another drug. We investigated the ability of phenylbutazone and oxyphenbutazone to mask or interfere with the detection of acidic drugs of high performance thin layer chromatography. Of 20 acidic drugs called "positive" since 1981 by laboratories affiliated with the Association of Official Racing Chemists, 16 did not comigrate with phenylbutazone or oxyphenbutazone and could not, therefore, be masked by these agents. Three medications (diclofenac, fenoprofen, ibuprofen) were potentially masked by phenylbutazone and one (sulindac) was potentially masked by oxyphenbutazone. These agents were therefore administered to horses to determine whether or not their metabolites would allow their detection. In each case, metabolites of these agents were detectable for at least 24 hr after drug administration and detection was not interfered with by phenylbutazone or oxyphenbutazone. These results suggest that these 20 acidic drugs should be readily detectable in postrace urines of horses in the presence of phenylbutazone either as the parent drug or by virtue of the easily distinguishable metabolites that each agent possesses. There is, therefore, no reason to believe that the agents tested in this study can be effectively masked or interfered with by phenylbutazone or its metabolites in equine urine.

Evidence for the fatty acid-induced heterogeneity of the N and B conformations of human serum albumin.

The influence of oleic acid on the interaction between albumin and warfarin, oxyphenbutazone or diazepam has been studied by circular dichroism and equilibrium dialysis. The pH dependences of the molar ellipticity of the drug-albumin complexes and of the free fraction of drug are completely changed by the presence of oleic acid. This phenomenon is attributed to an oleic acid-induced conformational change in both the neutral (N) and the basic (B) conformation of albumin, a change to which the warfarin-oxyphenbutazone binding area and the diazepam binding site is sensitive. The oleic acid-induced conformational states of albumin, the so-called N* and B* conformations, show binding properties that are different from the binding properties of the N and B conformations.

Vaginal contraceptive activity of hyaluronidase and cyclooxygenase (prostaglandin synthetase) inhibitors in the rabbit.

Seven inhibitors of the sperm enzyme hyaluronidase were tested at nonspermicidal concentrations for their vaginal contraceptive activity in rabbits. Five of these compounds are marketed antiinflammatory agents and the other two were synthesized. Most of the agents showed contraceptive activity. Of the antiinflammatory agents, phenylbutazone and oxyphenbutazone were particularly potent. Besides being hyaluronidase inhibitors, these compounds are also cyclooxygenase (prostaglandin synthetase) inhibitors; based on other in vitro data, their primary effect on spermatozoa is probably by the inhibition of that enzyme.

Effect on platelet aggregation of oral administration of 10 non-steroidal analgesics to humans.

Platelet aggregation was investigated in platelet-rich plasma from normal volunteers before and at various times after intake of 10 analgesic drugs. The drugs used were aspirin, piroxicam, naproxen, indomethacin, diclofenac, ibuprofen, diflunisal, paracetamol and oxyphenbutazone. Aggregation of the platelets was induced by adrenaline or ADP and the first and second waves of aggregation were evaluated. It was found that no drug exerted any effect on the first wave of aggregation. The second wave of aggregation was abolished by aspirin that produced a long-lasting effect for 5-8 d. Piroxicam also abolished the second wave of aggregation and this effect persisted on the 2 following d. Naproxen was normalized in half of the volunteers on the 2nd d. The inhibition caused by indomethacin and diclofenac was corrected on the 2nd d. Ibuprofen and diflunisal produced a definite but short-term effect. The effect of salicylic acid was weak. Paracematol and oxyphenbutazone did not affect platelet aggregation.