Novel approach to pro-drugs of lactones: water soluble imidate and ortho-ester derivatives of a furanone-based COX-2 selective inhibitor

Interest in water soluble COX-2 inhibitors that can be administered intravenously led to the development of novel pro-drugs of a furanone based COX-2 inhibitor 2. Transforming the lactone moiety of the furanone to an imidate or an ortho-ester with a hydrophilic, endogenous appendage resulted in water soluble pro-drugs that converted to the parent drug in vivo.     

Protective effect of a caspase inhibitor in models for cerebral ischemia in vitro and in vivo.

In primary neuronal-astrocyte cultures from mouse brain, ischemic conditions were simulated by combined oxygen-glucose deprival (OGD) for 2 hrs. This treatment resulted in near complete neuronal damage 24 hrs. later and was accompanied by DNA degradation and apoptotic nuclear morphology. Since caspases are key enzymes in the propagation and execution of programmed cell death, we evaluated the effect of the caspase inhibitor z-VAD-fmk. Damage following 2 hrs. OGD could be reduced by up to 56% with z-VAD-fmk (p<0.05). DNA-fragmentation and caspase activation has been also reported in an in vivo model of cerebral ischemia imitating human stroke. In this model the middle cerebral artery (MCA) is permanently occluded resulting in focal cerebral ischemia and subsequent infarction. Since z-VAD.fmk does not penetrate the blood-brain barrier it was applied intraventricularly as a bolus injection given 30 min. before MCA occlusion which was followed by 24 hrs. of infusion. Infarct volume was determined 48 hrs. after MCA occlusion by means of in vivo magnetic resonance imaging. Z-VAD.fmk dose dependently reduced infarct volume reaching a significant decrease of the cortical infarct by 45% when given as a 120 ng bolus followed by 40 ng/hr. infusion (p<0.05). In summary, our study supports the concept that caspase inhibitors are beneficial in brain ischemia.     

Cyclooxygenase-2 (COX-2) in acute myocardial infarction: cellular expression and use of selective COX-2 inhibitor

Our previous work has shown strong expression of COX-2 in the myocardium of patients with end-stage ischemic heart failure. The purpose of this study was to determine the cellular expression of this enzyme in the setting of acute myocardial infarction (AMI) and determine the role of COX-2 in experimental animals using a selective COX-2 inhibitor. Experimental AMI was induced in rats by ligating the left coronary artery. Animals were either treated with a selective COX-2 inhibitor (5 mg x kg(-1) x day(-1)) or vehicle. Three days after ligation, cardiac function was assessed and infarct size was determined. Myocardial specimens were immunostained with antiserum to COX-2. Plasma concentration of prostanoids was measured by enzyme immunoassay. There was strong expression of COX-2 in the myocytes, endocardium, vascular endothelial cells, and macrophages in the infarcted zone of the myocardium. In contrast, little expression was seen in the myocardium of control rats. Animals treated with the COX-2 inhibitor showed a significant improvement in left ventricular (LV) end-diastolic pressure (P < 0.05) and LV systolic pressure (P < 0.01), and a reduction in infarct size (P < 0.05). Inhibition of COX-2 significantly decreased plasma concentration of thromboxane B2 (P < 0.05); however, it did not affect 6-keto-prostaglandin F1alpha. Induction of COX-2 during AMI appears to contribute to myocardial injury, and treatment with the specific inhibitor of the enzyme ameliorated the course of the disease.     

Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia

ABSTRACT: BACKGROUND: Cerebral ischemia has been shown to induce activation of matrix metalloproteinases (MMPs), particularly MMP-9, which is associated with impairment of the neurovasculature, resulting in blood-brain barrier breakdown, hemorrhage and neurodegeneration. We previously reported that the thiirane inhibitor SB-3CT, which is selective for gelatinases (MMP-2 and 9), could antagonize neuronal apoptosis after transient focal cerebral ischemia. RESULTS: Here, we used a fibrin-rich clot to occlude the middle cerebral artery (MCA) and assessed the effects of SB-3CT on the neurovasculature. Results show that neurobehavioral deficits and infarct volumes induced by embolic ischemia are comparable to those induced by the filament-occluded transient MCA model. Confocal microscopy indicated embolus-blocked brain microvasculature and neuronal cell death. Post-ischemic SB-3CT treatment attenuated infarct volume, ameliorated neurobehavioral outcomes, and antagonized the increases in levels of proform and activated MMP-9. Embolic ischemia caused degradation of the neurovascular matrix component laminin and tight-junction protein ZO-1, contraction of pericytes, and loss of lectin-positive brain microvessels. Despite the presence of the embolus, SB-3CT mitigated these outcomes and reduced hemorrhagic volumes. Interestingly, SB-3CT treatment for seven days protected against neuronal laminin degradation and protected neurons from ischemic cell death. CONCLUSION: These results demonstrate considerable promise for the thiirane class of selective gelatinase inhibitors as potential therapeutic agents in stroke therapy.     

A double prodrug system for colon targeting of benzenesulfonamide COX-2 inhibitors

The design, synthesis and delivery potential of a new type of benzenesulfonamide cyclo-oxygenase-2 (COX-2) inhibitor prodrug is investigated using celecoxib. The approach involves a double prodrug that is activated first by azoreductases and then by cyclization triggering drug release. We studied the intramolecular aminolysis of the acylsulfonamide. The cyclization was surprisingly rapid at physiological pH and very fast at pH 5. The prodrug is activated specifically under conditions found in the colon but highly stable in the presence of human and rodent intestinal extracts. Finally, the prototype with celecoxib was transported much more slowly in the Caco-2 transepithelial model than the parent. The design therefore shows significant promise for the site specific delivery of benzenesulfonamide COX-2 inhibitors to the colon.     

Discovery of a potent and selective COX-2 inhibitor in the alkoxy lactone series with optimized metabolic profile

The COX-2 inhibitor DFP [5,5-dimethyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone] was found to have a long half-life in humans. Analogues have been characterized in order to optimize pharmacokinetics. This has lead to the discovery of 5(S)-(5-ethyl-5-methyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone analogue 11 a potent and selective COX-2 inhibitor which is metabolized to a greater extent than DFP upon incubation with rat and human hepatocytes, suggesting a shorter half-life in humans.     

青藤碱抑制人宫颈癌的研究

目的：研究青藤碱（sinomenine,SIN）对宫颈癌Hela细胞增殖的影响及其机制,为SIN在宫颈癌的预防和治疗上提供实验依据。方法：不同浓度SIN分别处理体外培养的人宫颈癌细胞系Hela细胞后,采用噻唑蓝（Mar）法检测处理24h、48h、72h后Hela细胞的增殖活性,流式细胞仪测定细胞周期和细胞凋亡。结果：1．0．1、0．2、0．4、0．625、1．25、2．5mmml／L SIN处理Hela细胞24h、48h、72h后,细胞增殖明显受到抑制,呈时间和剂量依赖性特点;2．流式细胞仪细胞周期分析表明,SIN处理组G1期细胞比例明显增加,S期细胞比例明显减少,两组比较有统计学意义;3．细胞凋亡分析表明,SIN处理组细胞凋亡率较对照组升高,呈时间和剂量依赖性特点;结论：SIN在体外能有效抑制宫颈癌细胞生长,其机制可能与其阻滞细胞周期、诱导细胞凋亡有关,SIN有望应用于宫颈癌的辅助治疗。     

Inhibition of ion channels and heart beat in Drosophila by selective COX-2 inhibitor SC-791

Recent findings suggest that modulation of ion channels might be implicated in some of the clinical effects of coxibs, selective inhibitors of cyclooxygenase-2 (COX-2). Celecoxib and its inactive analog 2,5-dimethyl-celecoxib, but not rofecoxib, can suppress or augment ionic currents and alter functioning of neurons and myocytes. To better understand these unexpected effects, we have recently investigated the mechanism of inhibition of human K(v)2.1 channels by a highly selective COX-2 inhibitor SC-791. In this study we have further explored the SC-791 action on ion channels and heartbeat in Drosophila, which lacks cyclooxygenases and thus can serve as a convenient model to study COX-2-independent mechanisms of coxibs. Using intracellular recordings in combination with a pharmacological approach and utilizing available Drosophila mutants, we found that SC-791 inhibited voltage-activated K(+) and L-type Ca(2+) channels in larval body-wall muscles and reduced heart rate in a concentration-dependent manner. Unlike celecoxib and several other K(+) channel blockers, SC-791 did not induce arrhythmia. Instead, application of SC-791 resulted in a dramatic slowing of contractions and, at higher concentrations, in progressively weaker contractions with gradual cessation of heartbeat. Isradipine, a selective blocker of L-type Ca(2+) channels, showed a similar pattern of heart arrest, though no prolongation of contractions was observed. Ryanodine was the only channel modulating compound of those tested additionally that was capable of slowing contractions. Like SC-791, ryanodine reduced heart rate without arrhythmia. However, it could not stop heartbeat completely even at 500 μM, the highest concentration used. The magnitude of heart rate reduction, when SC-791 and ryanodine were applied together, was smaller than expected for independent mechanisms, raising the possibility that SC-791 might be interfering with excitation-contraction coupling in Drosophila heart.     

Role of cytokines in experimentally induced lung cancer and chemoprevention by COX-2 selective inhibitor, etoricoxib

This study explored the role of pro- and anti-inflammatory cytokines in dimethyl benz(a)anthracene (DMBA)-induced lung cancer and its subsequent correction with a COX-2 inhibitory NSAID, etoricoxib. A single dose of DMBA (20?mg/kg body weight) in 0.9?% NaCl administered intratracheally was used to induce tumors in the rat lungs in 20?weeks. The study of pro-inflammatory cytokines like IL-1??, TNF-??, and IFN-?? revealed their upregulation by DMBA administration and restoration of their levels toward normal by the treatment with etoricoxib, while the anti-inflammatory cytokine IL-2 was found to be down-regulated with carcinogen administration and corrected with etoricoxib treatment. Apoptosis was studied by mitochondrial Bcl-2/Bax ratio and staining with fluorescent dyes acridine orange/ethidium bromide. The results showed a decreased apoptotic level with DMBA which was corrected with etoricoxib. Also, mitochondrial membrane potential was studied using JC-1 and rhodamine-123, which are membrane permeant fluorescent dyes, and generate information about cells at lower and higher mitochondrial membrane potential (???_M). The results showed the presence of maximum number of cells with higher ???_M in the DMBA group and their number was considerably lowered in the other three groups.     

The discovery of AZD5597, a potent imidazole pyrimidine amide CDK inhibitor suitable for intravenous dosing

The development of a novel series of imidazole pyrimidine amides as cyclin-dependent kinase (CDK) inhibitors is described. Optimisation of inhibitory potency against multiple CDK's (1, 2 and 9) resulted in imidazole pyrimidine amides with potent in vitro anti-proliferative effects against a range of cancer cell lines. Excellent physiochemical properties and large margins against inhibition of CYP isoforms and the hERG ion channel were achieved by modification of lipophilicity and amine basicity. A candidate with disease model activity in human cancer cell line xenografts and with suitable physiochemical and pharmacokinetic profiles for intravenous (i.v.) dosing was selected for further development as AZD5597.     

Targeting KSHV/HHV-8 latency with COX-2 selective inhibitor nimesulide: a potential chemotherapeutic modality for primary effusion lymphoma

The significance of inflammation in KSHV biology and tumorigenesis prompted us to examine the role of COX-2 in primary effusion lymphoma (PEL), an aggressive AIDS-linked KSHV-associated non-Hodgkin's lymphoma (NHL) using nimesulide, a well-known COX-2 specific NSAID. We demonstrate that (1) nimesulide is efficacious in inducing proliferation arrest in PEL (KSHV+/EBV-; BCBL-1 and BC-3, KSHV+/EBV+; JSC-1), EBV-infected (KSHV-/EBV+; Raji) and non-infected (KSHV-/EBV-; Akata, Loukes, Ramos, BJAB) high malignancy human Burkitt's lymphoma (BL) as well as KSHV-/EBV+ lymphoblastoid (LCL) cell lines; (2) nimesulide is selectively toxic to KSHV infected endothelial cells (TIVE-LTC) compared to TIVE and primary endothelial cells (HMVEC-d); (3) nimesulide reduced KSHV latent gene expression, disrupted p53-LANA-1 protein complexes, and activated the p53/p21 tumor-suppressor pathway; (4) COX-2 inhibition down-regulated cell survival kinases (p-Akt and p-GSK-3β), an angiogenic factor (VEGF-C), PEL defining genes (syndecan-1, aquaporin-3, and vitamin-D3 receptor) and cell cycle proteins such as cyclins E/A and cdc25C; (5) nimesulide induced sustained cell death and G1 arrest in BCBL-1 cells; (6) nimesulide substantially reduced the colony forming capacity of BCBL-1 cells. Overall, our studies provide a comprehensive molecular framework linking COX-2 with PEL pathogenesis and identify the chemotherapeutic potential of nimesulide in treating PEL.     

Structural basis for selective inhibition of COX-2 by nimesulide

Nimesulide 1 is a novel nonsteroidal antiinflammatory drug which inhibits the enzyme cyclooxygenase 2 (COX-2) more selectively than cyclooxygenase 1 (COX-1). Molecular modelling studies have been carried out on complexes of 1 with COX-1 and with mutants of COX-1 simulating COX-2. These indicate that the mutations I523V and S516A largely contribute to the selectivity. A comparative study with SC-558 2 has also been performed.     

Inducible COX-2 dominates over COX-1 in prostacyclin biosynthesis: mechanisms of COX-2 inhibitor risk to heart disease

AimOur aim is to understand the molecular mechanisms of the selective nonsteroidal anti-inflammatory drugs (NSAID), cyclooxygenase-2 (COX-2) inhibitors', higher “priority” to reduce synthesis of the vascular protector, prostacyclin (PGI2), compared to that of nonselective NSAIDs.Main methodsCOX-1 or COX-2 was co-expressed with PGI2 synthase (PGIS) in COS-7 cells. The Km and initial velocity (½t Vmax) of the coupling reaction between COX-1 and COX-2 to PGIS were established. The experiment was further confirmed by a kinetics study using hybrid enzymes linking COX-1 or COX-2 to PGIS. Finally, COX-1 or COX-2 and PGIS were respectively fused to red (RFP) and cyanic (CFP) fluorescence proteins, and co-expressed in cells. The distances between COXs and PGIS were compared by FRET.Key findingsThe Km for converting arachidonic acid (AA) to PGI2 by COX-2 coupled to PGIS is ~ 2.0 μM; however, it was 3-fold more (~ 6.0 μM) for COX-1 coupled to PGIS. The Km and ½t Vmax for COX-2 linked to PGIS were ~ 2.0 μM and 20 s, respectively, which were 2–5 folds faster than that of COX-1 linked to PGIS. The FRET study found that the distance between COX-2-RFP and PGIS–CFP is shorter than that between COX-1-RFP and PGIS–CFP.SignificanceThe study provided strong evidence suggesting that the low Km, faster ½t Vmax, and closer distance are the basis for COX-2 dominance over COX-1 (coupled to PGIS) in PGI2 synthesis, and further demonstrated the mechanisms of selective COX-2 inhibitors with higher potential to reduce synthesis of the vascular protector, PGI2.     

Assessment of the relative contribution of COX-1 and COX-2 isoforms to ischemia-induced oxidative damage and neurodegeneration following transient global cerebral ischemia

We investigated the relative contribution of COX-1 and/or COX-2 to oxidative damage, prostaglandin E2 (PGE2) production and hippocampal CA1 neuronal loss in a model of 5 min transient global cerebral ischemia in gerbils. Our results revealed a biphasic and significant increase in PGE2 levels after 2 and 24-48 h of reperfusion. The late increase in PGE2 levels (24 h) was more potently reduced by the highly selective COX-2 inhibitor rofecoxib (20 mg/kg) relative to the COX-1 inhibitor valeryl salicylate (20 mg/kg). The delayed rise in COX catalytic activity preceded the onset of histopathological changes in the CA1 subfield of the hippocampus. Post-ischemia treatment with rofecoxib (starting 6 h after restoration of blood flow) significantly reduced measures of oxidative damage (glutathione depletion and lipid peroxidation) seen at 48 h after the initial ischemic episode, indicating that the late increase in COX-2 activity is involved in the delayed occurrence of oxidative damage in the hippocampus after global ischemia. Interestingly, either selective inhibition of COX-2 with rofecoxib or inhibition of COX-1 with valeryl salicylate significantly increased the number of healthy neurons in the hippocampal CA1 sector even when the treatment began 6 h after ischemia. These results provide the first evidence that both COX isoforms are involved in the progression of neuronal damage following global cerebral ischemia, and have important implications for the potential therapeutic use of COX inhibitors in cerebral ischemia.     

The effect of intravenous tissue-type plasminogen activator in a rat model of embolic cerebral ischemia

Thrombolytic agents may be useful in the treatment of cerebral ischemia caused by arterial thrombosis or embolic occlusion. A trial of intravenous human tissue-type plasminogen activator (rt-PA) was carried out in seven male Sprague-Dawley rats subjected to embolic cerebral ischemia, with eight control animals. One-hour-old autogenous blood clot was injected into the internal carotid artery. A 30-minute infusion of 10 micrograms/kg/minute of rt-PA or saline followed. Areas of ischemia at two hours post-embolization were assessed by digital image processing of serial iodo-14C-antipyrine autoradiographic images. The volumes of "no-flow" (NF) and "low-flow" (LF) regions were calculated. One animal in each group suffered no detectable ischemia; the remainder had well-defined regions of middle and posterior cerebral artery ischemia. No animal sustained a hemorrhagic lesion. Treatment produced no noticeable effect on the patency of cervical vessels. Total NF and LF volumes were less for the treated group but did not reach statistical significance by t-test. In middle cerebral distribution sections, however, LF volume was significantly less (p less than 0.05) for treated animals (150 vs. 191 mm3), primarily due to a more significant decrease in LF volume in the anterior-middle cerebral overlap zone (47 vs. 90 mm3; p less than 0.025). Fibrinogen levels were not altered by drug treatment (p greater than 0.30).     

Resveratrol is a peroxidase-mediated inactivator of COX-1 but not COX-2: a mechanistic approach to the design of COX-1 selective agents

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a phytoalexin found in grapes that has anti-inflammatory, cardiovascular protective, and cancer chemopreventive properties. It has been shown to target prostaglandin H(2) synthase (COX)-1 and COX-2, which catalyze the first committed step in the synthesis of prostaglandins via sequential cyclooxygenase and peroxidase reactions. Resveratrol discriminates between both COX isoforms. It is a potent inhibitor of both catalytic activities of COX-1, the desired drug target for the prevention of cardiovascular disease, but only a weak inhibitor of the peroxidase activity of COX-2, the isoform target for nonsteroidal anti-inflammatory drugs. We have investigated the unique inhibitory properties of resveratrol. We find that it is a potent peroxidase-mediated mechanism-based inactivator of COX-1 only (k(inact) = 0.069 +/- 0.004 s(-1), K(i(inact)) = 1.52 +/- 0.15 microm), with a calculated partition ratio of 22. Inactivation of COX-1 was time- and concentration-dependent, it had an absolute requirement for a peroxide substrate, and it was accompanied by a concomitant oxidation of resveratrol. Resveratrol-inactivated COX-1 was devoid of both the cyclooxygenase and peroxidase activities, neither of which could be restored upon gel-filtration chromatography. Inactivation of COX-1 by [(3)H]resveratrol was not accompanied by stable covalent modification as evident by both SDS-PAGE and reverse phase-high performance liquid chromatography analysis. Structure activity relationships on methoxy-resveratrol analogs showed that the m-hydroquinone moiety was essential for irreversible inactivation of COX-1. We propose that resveratrol inactivates COX-1 by a "hit-and-run" mechanism, and offers a basis for the design of selective COX-1 inactivators that work through a mechanism-based event at the peroxidase active site.     

A general carbometalation, three component coupling strategy for the synthesis of alpha,beta-unsaturated gamma-sultines including thio-rofecoxib, a selective COX-2 inhibitor

Magnesium mediated carbometalation (Grignard addition) to appropriate propargyl alcohols to synthesize a cross-section of variably substituted alpha,beta-unsaturated gamma-sultines is described. Thio-rofecoxib, a selective COX-2 inhibitor (12), is synthesized by this method and its IC(50), microM COX-1 and COX-2 inhibition, and whole blood stability values reported.