26.80 mL = .02680 L Part 3: Preparation and Standardization of HCl Objective 3 The third objective of the lab was to determine the concentration of the HCl.
Vane’s success attracted many researchers to the area. Their investigations spread from aspirin to similar drugs that suppress pain and inflammation. By 1974, it was fairly well established that all NSAIDs act with similar mechanisms. They are all COX inhibitors.
Empirical observations yield dots of data. To connect them objectively depends on appropriate concepts and theories. The concept of COX-inhibition connects aspirin’s medicinal effects to prostaglandin and COX action. Due to the conceptual connection, these hitherto disparate pieces of knowledge become nexus in a network of relations. Furthermore, the network reveals other nexus and patterns that no one had dreamed of before. Therein lies the power of scientific theories to predict new phenomena, raise new questions, and indicate new areas for inquiry. That is why scientists highly value concepts and theories that correctly extract a set of relevant data and reveal their connections.
With the concept of COX-inhibition, knowledge about aspirin changes from mere empiricism to theory guided research. Whereas an empirical fact is specific to a particular phenomenon, a concept is general and potentially applicable to other phenomena. COX enzyme is present in many parts of the body, including unexpected places such as colon tumors. The conceptual framework of COX inhibition suggests links between aspirin and phenomena hitherto deemed unrelated, thus enabling scientists to ask significant questions and direct their research efforts. Basic scientists can use NSAIDs as tools to probe the physiological effects of COX, for instance in the formation of cancer. Pharmaceutical firms can use COX enzyme in test tubes to screen for promising drugs. A conceptual framework that explains phenomena by their underlying mechanisms is not a last word but a scientific breakthrough. A final word closes the door on exploration, a breakthrough opens up a frontier of research.
Enzymes are protein catalysts that speed up chemical reactions without being themselves used up in the reactions. An enzyme is a huge molecule with an active area that works somehow like a mold that accepts certain raw pieces and casts them into a final form. Imagine a mold that stamps a rod and a bowl into a spoon. Spoon production would be disrupted if someone throws a monkey range into the mold. Such a monkey range – an enzyme inhibitor – would make a desirable drug if it stops an enzyme from producing disease-inducing chemicals. Aspirin is an enzyme inhibitor. It suppresses the action of the enzyme COX, stops the production of prostaglandin, thus disrupting the pathways to pain, inflammation, elevated temperature, and stomach protection.
So far, aspirin research mainly marched from the top down, from organisms to tissues to enzymes. Now biochemistry rendezvoused with a branch of science marching from the bottom up, from atoms to enzymes. When two sciences meet, their concepts mesh and many more dots are connected to yield improved intelligence. Using X-ray crystallography and other technologies, molecular biologists were unraveling the molecular structures of genes and the enzymes encoded by the genes. They discovered in 1991 a novel gene that coded for an enzyme highly similar, but not identical, to the COX that was isolated from smooth muscles and widely studied. Soon molecular biologists established that COX enzyme has two forms, called COX1 and COX2. The two are coded by different genes and serve different physiological functions.
To understand the molecular basis for the antitumoureffects of aspirin and identify more effective alternatives, wepreviously synthesised a series of derivatives of the aspirinmolecule. These studies revealed that diaspirin (DiA) and fumaryldiaspirin (F-DiA) inhibit proliferation of CRC cell lines atsignificantly lower concentrations than aspirin (). To extend these studies and identifyfurther lead molecules, we synthesised an additional series ofaspirin derivatives ().Cytotoxicity (MTT) assays demonstrated that at 0.5 mM(pharmacologically relevant dose for aspirin), DiA (PN508) andF-DiA (PN517) and to an even greater extent, isopropylm-bromobenzoylsalicylate (PN529) reduced the viability of SW480 CRCcells (). To investigate thespecificity of compound toxicity in more detail, we tested thecapacity of DiA, F-DiA and PN529 to affect proliferation of anumber of established cell lines ( and ), controlling for any variabilitythat could arise from cell culture conditions through culturing allcells with DMEM as the basal medium. While we noted that culturingSW480 cells in their non-native medium (DMEM rather than L-15medium) reduced the sensitivity of the cells to the compoundstested, DiA and F-DiA in this assay system arguably showed amodicum of specificity towards the other CRC cell lines tested(HCT116 and LoVo), and given our finding that PN529 can inducenecrosis (), we focused ourattention on the anti-proliferative activity of DiA and F-DiA inmore detail and .
Ferric Chloride Test for Purity Chemical Reagents: .1M HCl, standardized
.1M NaOH, standardized
Phenolphthalein solution Step 1 Approximately .5g of the synthesized aspirin was weighed and placed into a 250mL Erlenmeyer flask.
The COX inhibition concept brought out many interesting questions. The NSAIDs are similar but not identical. They exhibit considerable variations in their effects. For instance, ibuprofen is easer on the stomach than aspirin. How can the variations be explained in terms of COX inhibition? This calls on basic science to uncover the detailed mechanisms by which the drugs interact with COX enzyme.
How does aspirin curb prostaglandin production? The many kinds of prostaglandin are synthesized by a host of complicated biochemical pathways. However, all pathways share a common stage facilitated by an enzyme called COX, whose action aspirin suppresses.
The major anti-proliferative effect of aspirin isrecognised to be the induction of apoptosis. However, the mechanismby which DiA and F-DiA act against CRC cells is as yet unknown. Tofurther understand this mechanism, we used Annexin V assays toinvestigate the effects of aspirin derivatives on apoptotic celldeath. We found that both compounds (3 mM) induced a significantincrease in the percentage of SW480 CRC cells undergoing apoptosis,compared to the carrier (DMSO)-treated controls (). This increase in apoptosis wasparalleled by a decrease in the percentage of viable cells,confirming the death-inducing capacities of the agents (data notshown). Time course studies revealed that 3 mM F-DiA mediated asignificant increase in apoptosis within 3 h of treatment (), while a more prolonged (>5 h)exposure was required before DiA-mediated apoptosis was evident(data not shown). In contrast to the diaspirin compounds, aspirinat 3 mM had minimal effect on cell growth/death after an 18-hexposure (data not shown), which is in keeping with previousstudies showing that 5 mM aspirin is required to induce detectableapoptosis of these cells within this time frame (). Taken together, these data indicatethat the diaspirin compounds induce apoptosis of CRC cells and thatthis occurs at a lower concentration than for aspirin and thatF-DiA is the more active of the compounds.