Cyclooxygenase Inhibitor in human diseases
Studies have shown that cyclooxygenase (COX) play an important role in our body. In this report we discuss how the inhibition of the COX-1 and COX-2 isoforms have on different diseases.
The paper begins with Serkan discussing the treatments of cancer with the use of cyclooxygenase inhibitors and the different mechanisms involved. This follows with a discussion for the treatment of Neuroblastomas by Nisha. The report is closed by Binal who covers the role of the inhibitors in inflammation in different diseases.
Cyclooxygenase, also referred to as COX, is an enzyme found in humans. Its key role is to convert arachidonic acid (arachidonate is first converted into an unstable endoperoxide intermediate by cyclooxygenase) into biological mediators called prostanoids; these include prostaglandins, prostacyclin and thromboxane.
Two forms of cyclooxygenase have been identified by scientists; COX-1 and COX-2. Although both have similar functions, they are used differently. COX-1 is a form that is considered as a constitutive enzyme. This means that it is an enzyme whose concentration in a cell is constant and is not influenced by substrate. Its primary function is to make prostaglandins available for homeostatic regulation. COX-2 is the form which can be induced and its main function is related to inflammation.
Both enzymes use arachidonic acid to make the same product, which is prostaglandin H2 (PGH2). Prostaglandins have various roles in the human body, for example, control cell growth and regulate inflammatory mediation. The precursors for prostanoids, e.g. Prostaglandin E2, have been found to enhance the growth of tumours and its invasiveness and protect against cell apoptosis. 
They have been found in many types of cancers, such as colorectal cancer, squamous cell carcinomas of the head and neck.
COX -2 inhibitors are usually tri-cyclic compounds, which have a central ring possessing a diaryl stilbene-like structure, with a sulfonyl group in the para position of the phenyl rings.2
The slight differences in the structure of COX-1 and COX-2 lead to their important pharmacological and biological differences. COX-1 has a smaller active site than COX-2. This difference is evident when looking at aspirin. During aspirin acetylation COX -1 is completely inhibited. However in the case of COX -2, the conversion of arachidonic acid is still possible.
The inhibition of this enzyme has many beneficial uses in the treatment of human diseases. Examples of this include providing relief from symptoms of pain and inflammation of tissues. However some serious side effects have also been identified, for example cases of cardiovascular disease and gastrointestinal complications.
ROLE of INHIBITORS IN CANCER
Cancer is a class of diseases which begins when cells from parts of the body begin to grow and divide uncontrollably resulting in the formation of abnormal cells. These cancerous cells can then invade and grow into surrounding tissues. With malignant tumours, the cancer cells can travel via the blood or lymph vessels and begin to grow and form new tumours in replacement of normal tissues in other parts of the body. This process is known as metastasis. Unlike malignant tumours, benign tumours do not always grow in an unlimited and aggressive manner, and do not invade surrounding tissues and metastasize.
The abnormal behaviour demonstrated by cancer cells are the result of a series of mutations in key regulatory genes. The cells become increasingly more abnormal as more genes become damaged. The genes that are in control of DNA repair (tumour suppressor genes) also become damaged, resulting in loss of normal function. This would include replicating DNA accurately, controlling the cell cycle, orientation within tissues and interacting with cells of the immune system. As a result, this leads to the progression of a tumour. DNA (gene expression) can be altered by; DNA mutations, translocations, gene amplification, inversions, duplications/deletions and aneuploidy.
Most cancers are caused by abnormalities in genetic material of transformed cells. This can be by chemical carcinogens which directly interact with DNA. Some chemicals become carcinogenic after being metabolised by cyctochrome P450 enzymes. Other carcinogens do not directly act on DNA but produce reactive oxygen species such as hydrogen peroxide, peroxynitrite and superoxide which all make significant DNA alterations to activate oncogenes or silence tumour suppressor genes.
Living a 'Western world' lifestyle is another cause of cancer. This would include a heavy consumption of alcohol, rich in fat diet and tobacco. These have been directly linked to cancers of lung, liver, pharynx; breast, prostate, colon, bladder, kidney and pancreas. Hereditary may also play a major part in some cases of cancer. If someone has a close family member diagnosed with prostate or breast cancer, it is very possible that these cancers will be found in that individual also. This is due to the inheritance of damaged DNA. Ethnicity can contribute to cancer, black Africans, Chinese and Native Americans have a higher risk of cancers of the prostate, nasopharynx and gall bladder.
Nonsteroidal anti-inflammatory drugs (NSAIDs) have become the most popular therapeutic agents in the treatment of pain, inflammation and fever since their discovery. However, NSAIDs may also have a pivotal role in chemoprevention; (the prevention, or delay of cancer development.) The clinical effectiveness of NSAIDs is caused by their ability to inhibit the cyclooxygenase (COX) enzymes. This enzyme has two isoforms which are COX-1 and COX-2. Even though both isoenzymes have the same catalytic activity on the same substrates, and are both about 60% homologous, they vary in regulation, distribution and expression.
Production of cancerous cells can be prevented by NSAIDs by inhibiting multiple steps in the carcinogenesis process. NSAIDs could influence certain cellular mechanisms such as angiogenesis, inflammation, cell differentiation and cell-turnover kinetics, all of which contribute towards carcinogenesis.10
By inhibiting COX enzymes, prostaglandin (PGs) synthesis in normal and inflamed tissues is reduced. PGs serve as autocrine or paracrine mediators to signal changes in normal physiology and pathologic conditions. They are short lived substances which are synthesised in a wide range of tissue types. PGs are involved in various functions around the body such as; nerve growth and development, immune responses and protection, blood clotting, bone metabolism, kidney function, ovulation and wound healing.>
In the mid-1980's, Needleman et al. proved the amount of COX enzyme found in inflamed tissues was significantly increased and was not dependent on the availability of arachidonic acid for the formation of PGs. This led to the discovery of the COX-2 enzyme which would be an attractive approach to produce a selective inhibitor of COX-2 in the treatment of inflammatory conditions without the toxicity side effects and inhibition of platelet function.
Chronic inflammation is a tumour promoter in almost all tissues that is associated with the pathogenesis of malignancies found in the gastrointestinal tract; this would include cancers of liver, stomach and colorectal. As NSAIDs have anti-inflammatory properties, it would be valid to assume that these agents would be able to inhibit inflammation, thus preventing cancer. NSAIDs impede inflammation and counter
cancer by the inhibition of the COX isoenzymes; however the catalytic activity of COX-2 is the most important isoform which is involved in this process.
COX-2 catalytic activity promotes carcinogenesis by producing activated carcinogens (cocarcinogen) and inflammatory mediators (eicosanoids). Another pathway in which NSAIDs can inhibit inflammation stimulated carcinogenesis is by preventing the activation of NF-kB, which controls the transcription of a range of pro-inflammatory cytokines, independently of COX inhibition.
Angiogenesis plays an important role in carcinogenesis and chronic inflammatory especially in the development of malignant carcinomas as well as pre-invasive benign neoplasia lesions such as colorectal adenomas. Angiogenesis is a vital process during embryonic development as it is the formation of new blood vessels from existing vasculature.14 The mechanism is as follows: -
1. Degradation of basement membrane proteolytic
2. Migration of endothelial cell
3. Proliferation of endothelial cell
4. Formation of lumen
5. Formation of new basement membrane
Due to the activity of angiogenesis in the production of cancerous cells, it is a feasible approach to use COX enzymes to target this area for inhibition. Of the two COX isoforms, the gene correlation of COX-2 has been linked with angiogenesis in benign premalignant cancers and tumours.14 COX-2 expressing cells secrete factors that induce actions of the angiogenesis process. The angiogenic mechanisms that get induced are the migration of endothelial cells and formation of the capillary tube. They are inhibited by the selective COX-2 inhibitor, NS-398, and non-selective inhibitor, ASA. The inhibition is concentration dependent between the two inhibitor sand is equivalent to the cancer cells in their reduced production of PG-E2. However, with colon cancer COX-2 expression, a variety of pro-angiogenic growth factors are also induced. These factors include bFGF-binding protein, VEGF, PDGF, TGF-b and endothelin-1. A combination of NS-398 and neutralizing antibodies prevent the expression of these factors. This suggests that these growth factors regulate COX-2 induced angiogenesis.
Some of the mechanisms which support the method of angiogenesis: -
1. Seed et al. reduced the growth of colon-26 cells in nude mice via an anti-angiogenic effect using a non-selective COX inhibitor, diclofenac. This had an anti-tumour effect.
2. Masferrer et al. studied various tumours from patients and detected the presence of COX-2 in the angiogenic vasculature and angiogenic blood vessels. They suggested COX-2 derived PGs induced newly formed blood vessels that sustained tumour cell growth. From this they proved that COX-2 inhibition in the presence of growth factor, FGF-2 had an anti-angiogeneic effect on the neovasculature.
3. Tsujii et al. found COX-1 activity in endothelial cells were directly associated with angiogenesis which could be used to treat tumours lacking COX-2 expression. The PGs produced by COX-1 in endothelial cells are important in regulating the genes required for endothelial tube formation. Inhibition of COX-1 by anti-sense oligonucleotide treatment significantly inhibited ETs-1 expression which is a transcription factor that has a role in angiogenesis. They suggested there were two mechanisms on how NSAIDs could inhibit angiogenesis: -
i. Inhibiting COX-1 activity in the endothelial cells
ii. Inhibiting COX-2 activity in colon carcinoma cells and down-regulating angiogenic factors
Another property of NSAIDs is the ability to induce apoptosis in a variety of cancers; this includes stomach, colon and prostate. Tsujii and DuBois reported that an increase in adhesion between epithelial cells over expressing COX-2 and extracellular matrix proteins led to a resistance of butyrate-induced apoptosis. However, with the addition of a non-specific COX inhibitor such as sulindac sulphide, these phenotypic changes can be reversed.16
In 1997, Elder et al. proved that this effect was not dependent on the over-expression of COX-2 and suggested that a selective COX-2 inhibitor, NS-389, had the ability to induce apoptosis which would lead to chemopreventative effects. This was later supported by Sawaoka et al. who found that in addition to NS-389, a non-selective inhibitor of COX-2, indomethacine, suppressed the growth and cell replication of gastric cancer by inducing apoptosis as well.16
Some of the mechanisms which support the method of apoptosis: -
1. Treatment of colon tumour cells which results in an increase in arachidonic acid that stimulates the production of a known death signal, ceramide, by activating neutral sphingomyelinase.
2. Tang et al. showed that the lipoxygenase pathway acts as a regulator of the apoptosis process.
3. Selective COX-2 inhibitors were used in down regulating the anti-apoptotic protein, Bcl-2 which resulted in making colon and prostate cancer cells sensitive to apoptosis.
4. A sulindac metabolite such as sulfone, cannot inhibit COX-2, but inhibits tumourgenesis by a COX-2 independent pathway during apoptosis signalling.
5. Lin et al. proposed cell survival was promoted by COX-2 through
up-regulating Mcl-1 by activating the P13K/Akt-pathway. The result of this showed that exposure to PG-E2 or over expression of COX-2 could lead to an increase of apoptosis in lung adenocarcinoma cells with the up-regulating Mcl-1 gene.
ROLE OF INHIBITORS IN NEUROBLASTOMAS
A neuroblastoma is a cancer of the nerve cells, neural crest cells. These particular cells are involved in the nervous systems development and other various tissues. They usually occur in one of the adrenal glands, but can also occur in the nerve tissues in the abdomen, pelvis, and chest and alongside the spinal cord in the neck. These cells are found in many solid tumours and are the most common type of cancer in paediatric patients. They are highly aggressive due their resistance to apoptosis and therefore have low-cure rates.
It has been found that treatment of neuroblastomas with COX -2 inhibitors have antiproliferative effects and cause apoptosis among these cells via the intrinsic mitochondrial pathway.
This is due to the fact that cyclooxygenase catalyses the conversion of arachidonic acid to prostaglandins, and it has been established that in neuroblastomas, the tumours express high levels of COX -2. In vitro studies have shown that when the COX -2 is inhibited and thereby increasing the abundance of arachidonic acid in the cells, cell death is induced. The effect of increased arachidonic acid concentration and administration of a COX -2 is further exaggerated when lipoxygenases are inhibited at the same time.22 These high levels of COX -2 are not seen in non-malignant tissues, which implies that it is this increased amount of COX -2 that is responsible for apoptosis resistance, promotion of cell proliferation and angiogenesis.
It has been found that diclofenac, a COX -1 and COX -2 inhibitor, brings about changes in the mitochondrial transmembrane potential, which causes the activation of enzymes (procaspase -9 and 3), followed by the induction of apoptosis. As a third enzyme (procaspase -8) is not activated it can be said that it is the NSAID that is causing the cell death.
Studies carried out in vivo using rats lacking thymus glands, have shown that tumour growths were significantly inhibited after treatment using diclofenac for two days, in comparison to rats that were untreated. The dosage also played a part in these studies; at 200mg/L tumour growth was completely inhibited for the first nine days, whereas at 250mg/L, the tumour growths were completely inhibited throughout the treatment period.
The same experiment was conducted on rats, but this time using celecoxib. The effect seen here was the same, tumour volumes were significantly reduced after four days and continues throughout the treatment. 4
These results were validated using immunohistochemistry of the tumours treated with diclofenac. They showed that there was an increase in the expression of cleaved caspase-3 (plays a vital role in the execution-phase of cell apoptosis.), than seen in untreated tumours, proving that it was in fact the diclofenac inducing the apoptosis of the neuroblastoma in vivo.
During the trials, 1H MRS studies were also conducted, as they provided clinical monitoring of tumour biochemistry. This is used to analyse intracellular lipids, such as polyunsaturated fatty acids (PUFA). It was found that there were increased levels of PUFA's and methylene groups of mobile lipids in cancer cells that were under-going treatment with inhibitors that induced call death among them. For this is can be deduced that NSAIDs can induce the accumulation of lipids (particularly PUFA's) in neuroblastoma cells. 22
This over expression of COX -2 is evident in a number of cancer types, for example, cervical carcinoma, prostate cancer and melanoma, which makes them potentially susceptible to COX -2 inhibition.
COX -2 inhibitors can be non-specific, such as aspirin and NSAIDs. Specific inhibitors, such as diclofenac or celeoxib are much newer to the world of science and therefore are still being further investigated as they have as yet only been mainly tested on tumours which are of epithelial origin.  However it has also been found that COX -2 expression increases after chemotherapy, so COX -2 inhibitor treatment, maybe used after the initial conventional therapies.
Increased concentrations of COX -2 have been found in areas of inflammation, however this was expected before testing, as some pro-inflammation agents like interleukin-1, tumour necrosis factor a and lipopolysaccharides are known to cause COX -2 expression. 22
Scientists have found many ways of trying to maintain cancers, chemotherapy and radiotherapy, even amputation and invasive surgery; however all of these have serious side effects and come with risks. In the search for alternative less toxic, possibly lower risk measures have been investigated; COX -2 inhibitors being one of them. They have provided results, which after further testing can be employed in real life situations, benefitting patients or varying ages greatly.
Another highly potent and selective COX -2 inhibitor (CAY10404) that maintains good analgesic and anti-inflammatory properties has been also investigated for its efficacy and the mechanism by which cell death is induced. Findings showed that CAY10404 did decrease the neuroblastomas cell growth in a dose-dependent manner. It was seen that cell proliferation decreased after 30M of CAY10404 was administered and at 115M 80% of cell growth was inhibited. This shows signs of apoptosis. The use of monoclonal antibodies detected the presence of activated caspase-3, which confirmed this further.
Role of Inhibitors in INFLAMMATION
When an injury occurs, the tissue becomes irritated or infected. Swelling and redness is usually observed when this occurs. When the inflammation pain persists for example, in rheumatoid arthritis and Alzheimer's disease it causes chronic inflammatory pain. The inflammation and its pain caused are initiated by the release of a large excess of diverse substances by the damaged tissue which activates the inflammatory process. 4 Some inflammatory mediators activate the nerve fibres involved and other mediators such as cytokines-Interleukin-1, growth factors and bacterial endotoxins stimulate the release via intracellular signalling of other mediators from the immune cells. It causes an induction of cyclooxygenase (COX-2) at the site of where the injury has occurred.
The pain is increased by COX-2 enzyme converting arachidonic acid to an unstable PGG2 and then further oxidized to a stable PGH2.1 Hence this arachidonic acid reaction leads to the synthesis of prostaglandins thromboxane, and prostacyclin. PGH2 can be further converted to a variety of eicosanoids by cyclooxygenase enzymes. Prostaglandins is also synthesised in small amounts by the other isoform of cyclooxygenase, COX-1. It is found in most tissues and regulates a normal homeostasis. 
The affect of non-selective inhibitor-NSAIDS
Non steroidal anti-inflammatory drugs (NSAIDs) are non selective inhibitors blocking both forms of cyclooxygenase. By blocking the enzyme COX-2 has been found to be a successful treatment in order to help ease the pain. The mechanism involved leads to reducing the amount of prostaglandins formed at the site of injury, hence reducing inflammation. However, when these NSAIDs inhibit COX-1, unwanted side effects can result such as gastrointestinal and renal side effects which has been discussed previously in the report.
NSAIDs are commonly used in human diseases such as pain relief and from severe pain relief caused by inflammation and joint diseases. Prostaglandins has an important role in inflammation as well as regulating physiological responses such as blood clotting, nerve growth and wound healing.3
NSAIDs such as indomethacin can be given which inhibit the COX-1 and COX-2 activity, these acts as a treatment in rheumatoid arthritis and osteoarthritis. Through the inhibition of the isoforms of cyclooxygenase, it reduces the level of prostaglandins as well as the inflamed tissues. However, long term use of NSAIDS can be limited due to its effect it has on the stomach by gastric toxicity and ulcer formation. Developments have been found by designing selective COX-2 inhibitors and blocking the production of prostaglandins and tissue inflammation. The advantage over using a selective inhibitor has shown no unwanted side effect such as gastric lesion and ulcer formation.
Response to Inflammation
Studies were conducted to analyse the inhibitory effects of NSAIDS. They were non selective for either forms of cyclooxygenase. The inhibition on COX-1 reduced synthesis of PG, however, developed an unwanted side effect of gastric lesion leading to ulceration.
In recent studies of selective inhibitors of COX-2: DUP-697 and NS-398 are anti-inflammatories which do not develop gastric ulceration. A mice study was conducted using COX-1 and COX-2 inhibitors such as indomethacin and the selective COX-2 inhibitors. It was carried out to show the biochemical efficacy of the non- selective and selective inhibitors. The PG analysis was determined by looking at the concentration present in both the pouch fluid where COX-2 is present and the stomach where COX-1 would be present. The study found that the NSAIDs inhibited the COX-1 enzyme and consequently it led to an increase in level of toxicity in the stomach. Also results showed there was a reduction of PGs production in the stomach was found due to the inhibition of the COX-1 enzyme. The selective inhibitors showed no affect on COX-1 enzyme in mice therefore PGs were formed and there were no signs of gastric ulceration. 4
Role of COX-2 and inhibitors in Alzheimer's disease
Alzheimer's disease is a neurological disorder of the brain and features of this disease have been found to be genetically inherited which are linked to the mutation on mitochondrial chromosomes. The disease causes memory loss and cognition due to the deposition of amyloid fibrils and nerofibrilary tangles within the brain.
Inflammation in Alzheimer's disease is regulated by NSAIDs which studies have found to prolong the clinical expression of the disease. The NSAIDs act by inhibiting the enzyme COX-2 which is responsible for synthesing prostaglandins from arachiodonic acid. PG in the brain is considered to be important and is present within limbic system.  Studies show that excitotoxic lesion in Alzheimer's stimulate an increase in release of COX-2 enzymes. Studies showed that 80% of COX-2 was present in the frontal cortex. The anti-inflammatory non- steroidal drugs target COX-2 enzyme.
Role of COX-2 and inhibitors in Arthritis
Animal studies of inflammatory arthritis have shown that COX-2 enzyme has been responsible for the increase in production of prostaglandins which is seen in the inflamed joint tissues. The increase of the prostaglandins has taken place in the affected cartilage in osteoarthritis and synovial tissue in rheumatoid arthritis. The inflammation mediators such cytokines cause an induction of COX-2 at the site of inflammation. Synovial tissues of patients were found to have low amount of COX-2 enzyme compared to the patients with osteoarthritis cartilage which showed large amounts of COX-2 and prostaglandins. A possible reason for this difference is that nitric oxide is an inflammatory modulator which regulates the production of prostaglandins in osteoarthritis but not present in the synovial tissue.3 In a rat arthritis study, inflammation was reduced by pharmalogical inhibition of COX-2 activity. A selective COX-2 inhibitor, SC-58125 was given to inhibit COX-2 without affecting COX-1 which decreased the level of prostaglandins hence reduced the inflammation of synovium.
SIDE EFFECTS OF NSAIDS AND CYCLOOXYGENASE INHIBITORS
Cardiovascular disease or heart and circulatory disease as it also known as, group together a number of conditions, such as coronary heart disease, angina, heart attack and strokes.
It is caused by the deposit of fat on the walls of the coronary arteries, causing them to narrow. Thereby making it difficult for the artery to supply the heart muscle with blood and vital oxygen. The correct medical term being atherosclerosis. Over time the narrowing of the artery can severely reduce the flow of blood to your heart, particularly when exerting yourself physically, causing pain and discomfort in the chest, also referred to as angina. If the fatty deposit breaks detaches itself from the artery wall, a blood clot may form, which could in turn block an artery, thereby starving the heart muscle completely of blood and oxygen, resulting in a heart attack.
COX -2 inhibitors can be used in the treatment of myocardial injury, as they are useful inflammatory mediators.
NSAIDS, which are non steroidal anti-inflammatory drugs, are used as a preventative measure against cardiovascular disease. These types of drugs evolved from the discovery of a common analgesic called aspirin (acetylsalicylic acid).
Studies have found that vascular endothelial cells and smooth muscle cells have COX-1, and that prostacyclin which is formed via COX-1 has an important role in blood flow, blood pressure and anti-aggregation of platelets, i.e. thinning of the blood. However, a recent investigation has shown that prostacyclin in vascular cells is produced by COX-2 as well as COX-1 under both physiological and pathological conditions.
In patients that were treated with a COX-2 specific inhibitor, found that their levels of urinary protacyclin metabolites had decreased, without affecting the thromboxane metabolite concentration. Treatment of volunteers with a COX-2 specific inhibitor decreased the levels of urinary prostacyclin metabolites without affecting thromboxane A2 metabolites. However, the NSAID, indomethacin, decreased metabolites of both prostacyclin and thromboxane. This led them to believe that the risk of CHD would be increased with COX-2 inhibitors.4
However recent research has indicated that possible side effects of drugs such as celeoxib and rofecoxib (which has been voluntarily withdrawn from worldwide sales since 2004) may actually increase the risk of heart attacks and strokes as prostaglandins are involved in regulation of blood pressure.
Studies have found that patients receiving high doses of rofecoxib (>25mg) actually largely increases the rate of serious coronary heart diseases, compared to those who take a lower dosage. These effects where not seen when celeoxib was administered to patients. 
These findings indicate that prescribing cyclooxygenase inhibitors can have some serious side effects if not prescribed according to individual patient needs.
With the use of NSAIDs, it has been found that patients have suffered from gastric ulcers (due to COX -1 inhibition), however COX -2 inhibitors which are selective actually reduce ulceration. Although these findings are promising, sufficient studies have not been carried out to give this data clinical significance.  The drug in question in this particular study was naproxen group. Results showed that 72% of patients suffered from gastric erosion or ulcers, compared with those using celecobix. It was endoscopic analysis that provided data for these findings in conjunction with the lack of platelet effect. However data does not indicate that there is a significant reduction in all GI symptoms, such as nausea, dyspepsia and abdominal pain; with specific COX -2 inhibitors.
Although these side effects are evident, they only become a problem with the dose at which the drug is administered. At low doses, most NSAIDs are relatively safe and side effects are not as common as they would be should the dose be increased. 
COX-1 also plays a role in vasodilatation in the present of contractile conditions in both the stomach and kidneys. During changes in blood flow, the kidney is stimulated to release mediators to maintain blood pressure by vasodilatation. COX-1 is present in the vasculature, glomeruli and collecting ducts of kidneys. The enzyme is important when prostaglandins are being synthesised as it maintains physiological functions such as glomerular filtration rate. 3 The NSAIDS inhibition of COX enzymatic activity affect the physiological conditions fail to respond and lead to renal damage .As well as this unwanted side effect, the production of prostaglandins is inhibited which leads to cell death.
To conclude cyclooxygenase have been found in many studies to play a vital part in human diseases. Selective and non-selective inhibitors of cyclooxygenase have been used to help prevent the symptoms such as inflammation. However, they have shown negative effects such as gastrointestinal complication, cardiovascular effects and renal damage. Therefore more studies into selective COX-2 inhibitors is required in human trails to fully determine its efficacy.
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