Kebreab Ghebreselasie

Chemical stability of 2-arachidonylglycerol under biological conditions.

Recent evidence indicates that 2-arachidonylglycerol (2-AG) is a potent and specific ligand for the central and peripheral cannabinoid receptors. Therefore, the chemical stability of this molecule under biological conditions is of interest. A method for the isolation and detection of 2-AG using HPLC with evaporative light scattering detection is described. The method provides an extraction recovery from aqueous media of 78%, and a limit of detection of 60 ng on column. Incubation of 2-AG in culture medium or biological buffers indicated that it is stable to oxidation and ester hydrolysis for up to 6 h at 37 degrees C. However, gradual disappearance of the compound was noted due to adherence to glass and plastic surfaces. During incubation in RPMI culture medium, 2-AG rearranged to 1(3)-arachidonylglycerol (1(3)-AG) in a first order process with a half-life of 10 min in the absence of serum and 2.3 min in the presence of 10% fetal calf serum. Further studies indicated that the acyl migration reaction is base catalyzed (k(cat)=78,000/min M), and that the reaction is affected slightly by changes in buffer (Tris) concentration and not at all by changes in ionic strength. The results indicate that 2-AG is readily converted to 1(3)-AG under conditions commonly used to study receptor-ligand interactions, findings that have significant implications for the interpretation of relative ligand potency between the two isomers.

Fluorinated Cyclooxygenase-2 Inhibitors as Agents in PET Imaging of Inflammation and Cancer

COX-2 is a major contributor to the inflammatory response and cancer progression so it is an important target for prevention and therapy. COX-2 is absent or expressed at low levels in most epithelial cells but is found at high levels in inflammatory lesions, and many premalignant and malignant tumors. Thus, it is an attractive target for molecular imaging. We report a series of novel fluorinated imaging agents, derived from indomethacin or celecoxib that selectively inhibit COX-2. The most promising lead, compound 7, was a fluorinated derivative of celecoxib. Kinetic analysis revealed that this fluorinated compound is a slow, tight-binding inhibitor of COX-2 and exhibits minimal inhibitory activity against COX-1. Efficient incorporation of 18F into compound 7 by radiochemical synthesis and intravenous injection provided sufficient signal for in vivo positron emission tomography (PET) imaging. Selective uptake of 18F-7 was observed in inflamed rat paws compared with the noninflamed contralateral paws and uptake was blocked by pretreatment with the COX-2 inhibitor, celecoxib. Uptake of 18F-7 was not observed when inflammation was induced in COX-2–null mice. In nude mice bearing both a COX-2–expressing human tumor xenograft (1483) and a COX-2–negative xenograft (HCT116), 18F-7 selectively accumulated in the COX-2–expressing tumor. Accumulation was blocked by pretreatment of the animals with celecoxib. The in vitro and in vivo properties of compound 7 suggest it will be a useful probe for early detection of cancer and for evaluation of the COX-2 status of premalignant and malignant tumors. Cancer Prev Res; 4(10); 1536–45. ©2011 AACR.

Design, Synthesis, and Structure–Activity Relationship Studies of Fluorescent Inhibitors of Cycloxygenase-2 as Targeted Optical Imaging Agents

Cycloxygenase-2 (COX-2) is an attractive target for molecular imaging because it is an inducible enzyme that is expressed in response to inflammatory and proliferative stimuli. Recently, we reported that conjugation of indomethacin with carboxy-X-rhodamine dyes results in the formation of effective, targeted, optical imaging agents able to detect COX-2 in inflammatory tissues and premalignant and malignant tumors (Uddin et al. Cancer Res. 2010, 70, 3618–3627). The present paper summarizes the details of the structure–activity relationship (SAR) studies performed for lead optimization of these dyes. A wide range of fluorescent conjugates were designed and synthesized, and each of them was tested for the ability to selectively inhibit COX-2 as the purified protein and in human cancer cells. The SAR study revealed that indomethacin conjugates are the best COX-2-targeted agents compared to the other carboxylic acid-containing nonsteroidal anti-inflammatory drugs (NSAIDs) or COX-2-selective inhibitors (COXIBs). An n-butyldiamide linker is optimal for tethering bulky fluorescent functionalities onto the NSAID or COXIB cores. The activity of conjugates also depends on the size, shape, and electronic properties of the organic fluorophores. These reagents are taken up by COX-2-expressing cells in culture, and the uptake is blocked by pretreatment with a COX inhibitor. In in vivo settings, these reagents become highly enriched in COX-2-expressing tumors compared to surrounding normal tissue, and they accumulate selectively in COX-2-expressing tumors as compared with COX-2-negative tumors grown in mice. Thus, COX-2-targeted fluorescent inhibitors are useful for preclinical and clinical detection of lesions containing elevated levels of COX-2.

Conjugation of cisplatin analogues and cyclooxygenase inhibitors to overcome cisplatin resistance.

Cyclooxygenase (COX) is an enzyme involved in tumorigenesis and is associated with tumor cell resistance against platinum‐based antitumor drugs. Cisplatin analogues were conjugated with COX inhibitors (indomethacin, ibuprofen) to study the synergistic effects that were previously observed in combination treatments. The conjugates ensure concerted transport of both drugs into cells, and subsequent intracellular cleavage enables a dual‐action mode. Whereas the platinum(II) complexes showed cytotoxicities similar to those of cisplatin, the platinum(IV) conjugates revealed highly increased cytotoxic activities and were able to completely overcome cisplatin‐related resistance. Although some of the complexes are potent COX inhibitors, the conjugates appear to execute their cytotoxic action via COX‐independent mechanisms. Instead, the increased lipophilicity and kinetic inertness of the conjugates seem to facilitate cellular accumulation of the platinum drugs and thus improve the efficacy of the antitumor agents. These conjugates are important tools for the elucidation of the direct influence of COX inhibitors on platinum‐based anticancer drugs in tumor cells.

Cyclooxygenase-1-Selective Inhibitors Based on the (E)-2′-Des-methyl-sulindac Sulfide Scaffold

Prostaglandins (PGs) are powerful lipid mediators in many physiological and pathophysiological responses. They are produced by oxidation of arachidonic acid (AA) by cyclooxygenases (COX-1 and COX-2) followed by metabolism of endoperoxide intermediates by terminal PG synthases. PG biosynthesis is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). Specific inhibition of COX-2 has been extensively investigated, but relatively few COX-1-selective inhibitors have been described. Recent reports of a possible contribution of COX-1 in analgesia, neuroinflammation, or carcinogenesis suggest that COX-1 is a potential therapeutic target. We designed, synthesized, and evaluated a series of (E)-2′-des-methyl-sulindac sulfide (E-DMSS) analogues for inhibition of COX-1. Several potent and selective inhibitors were discovered, and the most promising compounds were active against COX-1 in intact ovarian carcinoma cells (OVCAR-3). The compounds inhibited tumor cell proliferation but only at concentrations >100-fold higher than the concentrations that inhibit COX-1 activity. E-DMSS analogues may be useful probes of COX-1 biology in vivo and promising leads for COX-1-targeted therapeutic agents.

Oxicams Bind in a Novel Mode to the Cyclooxygenase Active Site via a Two-water-mediated H-bonding Network

Background: The oxicams are anti-inflammatory drugs targeting the cyclooxygenase enzymes. Results: Crystal complexes of mCOX-2·isoxicam, mCOX-2·meloxicam, and oCOX-1·meloxicam are solved. Conclusion: Oxicams bind to the cyclooxygenase active sites in a novel mode. Significance: The first structural description of cyclooxygenase-oxicam complexes reveal a new binding pocket of inhibitors to cyclooxygenases. Oxicams are widely used nonsteroidal anti-inflammatory drugs (NSAIDs), but little is known about the molecular basis of the interaction with their target enzymes, the cyclooxygenases (COX). Isoxicam is a nonselective inhibitor of COX-1 and COX-2 whereas meloxicam displays some selectivity for COX-2. Here we report crystal complexes of COX-2 with isoxicam and meloxicam at 2.0 and 2.45 angstroms, respectively, and a crystal complex of COX-1 with meloxicam at 2.4 angstroms. These structures reveal that the oxicams bind to the active site of COX-2 using a binding pose not seen with other NSAIDs through two highly coordinated water molecules. The 4-hydroxyl group on the thiazine ring partners with Ser-530 via hydrogen bonding, and the heteroatom of the carboxamide ring of the oxicam scaffold interacts with Tyr-385 and Ser-530 through a highly coordinated water molecule. The nitrogen atom of the thiazine and the oxygen atom of the carboxamide bind to Arg-120 and Tyr-355 via another highly ordered water molecule. The rotation of Leu-531 in the structure opens a novel binding pocket, which is not utilized for the binding of other NSAIDs. In addition, a detailed study of meloxicam·COX-2 interactions revealed that mutation of Val-434 to Ile significantly reduces inhibition by meloxicam due to subtle changes around Phe-518, giving rise to the preferential inhibition of COX-2 over COX-1.

[123I]-Celecoxib Analogues as SPECT Tracers of Cyclooxygenase-2 in Inflammation

We report the synthesis and evaluation of a series of iodinated celecoxib analogues as cyclooxygenase-2 (COX-2)-targeted single photon emission computerized tomography (SPECT) imaging agents for the detection of inflammation. The structure−activity relationship identified 5-(4-iodophenyl)-1-{4-(methylsulfonyl)phenyl}-3-(trifluoromethyl)-1H-pyrazole (8) as a promising compound with IC50 values of 0.05 μM against purified COX-2 and 0.03 μM against COX-2 in activated macrophages. The arylstannane of 8 undergoes facile radio-[123I]-iodination upon treatment with Na123I/NaI and chloramine T using an EtOAc/H2O two-phase system. The [123I]-8 was produced in a radiochemical yield of 85% and a radiochemical purity of 99%. In vivo SPECT imaging demonstrated that the radiotracer was taken up by inflamed rat paws with an average 1.7-fold enrichment over contralateral noninflamed paws. This study suggests that conversion of celecoxib into its isomeric iodo-[123I]-analogues is a useful approach for generating novel and efficacious agents for COX-2-targeted SPECT imaging of inflammation.

nido‐Dicarbaborate Induces Potent and Selective Inhibition of Cyclooxygenase‐2

Carbaboranes are increasingly studied as pharmacophores, particularly as replacements for aromatic systems. However, especially ortho‐carbaborane is prone to degradation of the cluster, which hampers biological application. This study demonstrates that deboronation of the cluster may not only lead to a more active analogue, but can also improve the solubility and stability of a carbaborane‐containing inhibitor. Notably, introduction of a nido‐dicarbaborate cluster into the cyclooxygenase (COX) inhibitor indomethacin results in remarkably increased inhibitory potency and selectivity for COX‐2 relative to the respective phenyl analogue. The first crystal structure of a carbaborane‐containing inhibitor bound to COX‐2 further reveals a novel binding mode for the inhibitor that is strikingly different from that of indomethacin. These results indicate that nido‐dicarbaborate is a promising pharmacophore that exhibits properties which are also highly beneficial for its introduction into other inhibitor classes.

PET radiotracer [18F]-P6 selectively targeting COX-1 as a novel biomarker in ovarian cancer: Preliminary investigation

Cyclooxygenase-1 (COX-1), but not COX-2, is expressed at high levels in the early stages of human epithelial ovarian cancer where it seems to play a key role in cancer onset and progression. As a consequence, COX-1 is an ideal biomarker for early ovarian cancer detection. A series of novel fluorinated COX-1-targeted imaging agents derived from P6 was developed by using a highly selective COX-1 inhibitor as a lead compound. Among these new compounds, designed by structural modification of P6, 3-(5-chlorofuran-2-yl)-5-(fluoromethyl)-4-phenylisoxazole ([(18/19)F]-P6) is the most promising derivative [IC50 = 2.0 μM (purified oCOX-1) and 1.37 μM (hOVCAR-3 cell COX-1)]. Its tosylate precursor was also prepared and, a method for radio[(18)F]chemistry was developed and optimized. The radiochemistry was carried out using a carrier-free K(18)F/Kryptofix 2.2.2 complex, that afforded [(18)F]-P6 in good radiochemical yield (18%) and high purity (>95%). In vivo PET/CT imaging data showed that the radiotracer [(18)F]-P6 was selectively taken up by COX-1-expressing ovarian carcinoma (OVCAR 3) tumor xenografts as compared with the normal leg muscle. Our results suggest that [(18)F]-P6 might be an useful radiotracer in preclinical and clinical settings for in vivo PET-CT imaging of tissues that express elevated levels of COX-1.

Design of Fluorine-Containing 3,4-Diarylfuran-2(5H)‑ones as Selective COX‑1 Inhibitors

We report the design and synthesis of fluorine-containing cyclooxygenase-1 (COX-1)-selective inhibitors to serve as prototypes for the development of a COX-1-targeted imaging agent. Deletion of the SO2CH3 group of rofecoxib switches the compound from a COX-2- to a COX-1-selective inhibitor, providing a 3,4-diarylfuran-2(5H)-one scaffold for structure-activity relationship studies of COX-1 inhibition. A wide range of fluorine-containing 3,4-diarylfuran-2(5H)-ones were designed, synthesized, and tested for their ability to selectively inhibit COX-1 in purified protein and human cancer cell assays. Compounds containing a fluoro-substituent on the C-3 phenyl ring and a methoxy-substituent on the C-4 phenyl ring of the 3,4-diarylfuran-2(5H)-one scaffold were the best COX-1-selective agents of those evaluated, exhibiting IC50s in the submicromolar range. These compounds provide the foundation for development of an agent to facilitate radiologic imaging of ovarian cancer expressing elevated levels of COX-1.