Synthesis of Novel Small Molecule LTA4H Enzyme Activators for COPD

Suzie Bae
Thomas Jefferson High School for Science and Technology

Abstract

Chronic obstructive pulmonary disease (COPD) is a progressive disease that leads to reduced airflow to the lungs due to persistent inflammation. The leukotriene A 4 hydrolase (LTA 4 H) enzyme is a bifunctional enzyme that catalyzes hydrolysis of leukotriene A 4 to leukotriene B 4 (epoxy hydrolase activity) as well as hydrolysis of  the N-terminus of tripeptides such as Pro-Gly- Pro (aminopeptidase (AP) activity). The hydrolysis of LTA 4 to LTB 4 is a pro-inflammatory pathway, and hydrolysis of Pro-Gly-Pro is an anti-inflammatory pathway. The small molecule 4-MDM selectively activates the LTA 4 H enzyme for AP activity.  However, 4-MDM suffers from undesirable physicochemical properties such as poor water solubility and at room temperature the compound exists as an oil. We hypothesized that introducing more oxygen atoms in the molecule could provide new compounds with improved properties, but preserve the activation properties of 4-MDM on the LTA 4 H AP activity. Acetic anhydride was reacted with 4-benzylphenol in the presence of pyridine in dichloromethane to afford the acetylated product. The compound was purified by column chromatography and characterized by NMR and GC/MS analysis. Enzyme assays show the compound, 4-AcDM, with increased oxygen atom content activated the LTA 4 H AP activity.

Introduction

Chronic obstructive pulmonary disease (COPD) is a progressive disease, primarily caused by cigarette smoking, that slowly and gradually makes breathing difficult [1]. Currently, COPD is the third leading cause of death in the United States [2], and resulted in 134,676 deaths in 2010 [2]. Symptoms of COPD include coughing, wheezing, and expectoration [1]. Emphysema is a sub-type of COPD that involves irreversible damage to the alveoli sacs [1]. Alveoli sacs are air sacs within the lungs at which oxygen and carbon dioxide are exchanged in blood. In emphysematous patients, the alveoli sacs expand due to inflammation [1]. Even when individuals quit smoking, they usually do not regain lung capacity [1]. For emphysematous patients, the process is irreversible and progressive. Emphysema involves the destruction of the alveolar walls in the alveoli sacs when alveolar macrophages release their proteases. The macrophages are important for normal lung function because proteases from the macrophages digest foreign material. However, under the persistent inflammation associated with emphysema, the proteases are harmful because they damage alveolar tissue and create emphysematous lesions [3]. Emphysema is also marked by a loss of elasticity that leads to narrowing of the thorax, contributing to greater airflow limitation [3]. As a result, elastin, an elastic protein in connective tissue, is destroyed [3]. Since tissue destruction in emphysema is permanent, the best solution to combating the disease is prevention before onset, through smoking cessation as early as possible [3].

The leukotriene A4 hydrolase (LTA 4 H) enzyme has two functions closely associated with pulmonary emphysema. The LTA 4 H enzyme is a bifunctional enzyme with both aminopeptidase (AP) and epoxy hydrolase (EH) activities. The EH activity participates in a pro-inflammatory response by converting leukotriene A4 (LTA 4) to leukotriene B4 (LTB 4). The LTB 4 metabolite signals neutrophil and macrophage chemotaxis, which results in alveolar destruction when the macrophages release their proteases. The LTA 4 H AP activity degrades the N-terminus of peptides of small tripeptides such as proline-glycine- proline (PGP). PGP was found to participate in inflammatory responses by inducing neutrophil chemotaxis. However, Paige et al. determined that the role of the LTA 4 H aminopeptidase in the pathogenesis of emphysema involves LTA 4 H-mediated hydrolysis of PGP and clearance of the neutrophil infiltration, which is an anti-inflammatory response. The proof-of-concept was shown using 4-MDM, a pharmaceutical agent designed to selectively augment the AP activity of the LTA 4 H enzyme. Cigarette smoke (CS) exposure in the absence of 4-MDM leads to CS-induced emphysema presumably due to suppression of LTA 4 HAP activity. However, treatment with 4-MDM rescues the lungs from emphysema by restoring the LTA 4 HAP activity, which eventually results in a protective anti-inflammatory response [4].

The LTA4H enzyme pathway has been studied as a target for emphysema because of its role in the synthesis of LTB 4 . Shin et al. reported that the metabolite LTB 4 is a neutrophil chemo-attractant and therefore linked to several inflammatory diseases, including cystic fibrosis, sepsis, and emphysematous COPD [10] . Further, it was found that inhibition of LTB 4 showed significant beneficial effects in suppressing emphysema. Although previous research efforts focused solely on the inhibition of LTB 4 as a treatment option, Oliveira et al.’s work suggested that the anti-inflammatory AP activity of LTA 4 H enzyme could be essential to future emphysema drug discovery efforts. In their drug synthesis, Oliveira et al. used the knowledge that 4-methoxyphenoxybenzene increases the AP activity of the LTA 4 H enzyme. On the basis of Lai group’s model of 4-methoxyphenoxybenzene, Oliveira et al. concluded that the central oxygen atom of the drug was not involved in binding, and likely was responsible for decomposition to toxic drug metabolites in animal models. Therefore, 4-MDM was designed by exchanging the ether linkage of 4-methoxyphenoxybenzene to a methylene bridge to the two aryl groups. The compound 4-methoxydiphenylmethane (4-MDM) showed increased stability. In a murine model of pulmonary emphysema, 4-MDM resulted in no observable toxicity in the mice over the entire study. Oliveira et al’s research is significant because of its focus on the lesser studied AP activity of the LTA 4 H enzyme as an improved strategy for treating inflammation associated with emphysema [5].

Based on the information from previous literature, we conducted our study on a pathway for which research was lacking, namely the LTA 4 HAP pathway. All previous drug discovery efforts targeting the LTA 4 H enzyme have focused on the EH pathway. Therefore, we synthesized 4-AcDM as a new lead compound with enhanced physicochemical properties. In place of the methoxy group, an acetyl group at the end of the compound was used in order to impart a reduced log P partition coefficient and increased expected water solubility [5]. Since the EH pathway of the LTA 4 H enzyme has been largely ineffective for emphysema treatment, likely due to its multiple mechanisms that promote inflammation, we focused our efforts on the AP pathway instead [5]. Existing drugs have focused solely on treating the symptoms after emphysema has already manifested itself. The problem lies in that these drugs do not halt the infiltration of inflammatory cells, which results in tissue damage. Our drug speeds up the clearance of inflammation, which we believe will be effective because we are targeting emphysema from its onset.

The EH activity of the LTA 4 H enzyme is responsible for the pro-inflammatory response to emphysema and the AP activity of the enzyme is responsible for the anti-inflammatory response. Inflammation can be initiated by the hydrolysis of leukotriene A4 to leukotriene B4, which is mediated by the LTA 4 H EH activity. However, clearance of inflammation is promoted by hydrolysis of the tripeptide proline-glycine- proline (PGP) by the LTA 4 H AP activity [4]. Our research question was how do we synthesize a compound that will target the AP pathway of the LTA4H enzyme? The hypothesis was that the synthesis of the compound, 4-AcDM, would be efficient in accelerating the rate of PGP hydrolysis, which is expected to correlate with an anti-inflammatory response in vivo. The hypothesis was developed upon the prediction that the replacement of methyl group from the already known compound 4-MDM with an acetyl group would create greater drug stability and increased solubility. The product to be developed was 4-AcDM, which was synthesized by acetylation of 4-benzylphenol in dichloromethane solvent in the presence of pyridine and acetic anhydride.

Emphysema is a disease worthy of study, as effective treatments to halt or even reverse the disease have yet to be identified. Regarding treatment for emphysema, solutions have focused on the use of leukotriene A 4 inhibitors, but the development of leukotriene A 4 aminopeptidase activators is limited [5]. Currently, treatments for emphysema are concentrated almost exclusively on lessening symptoms rather than extending the lifespan of those living with the disease and clearing inflammation as a whole. In 2013, 17.8% of adults residing in the U.S. smoked cigarettes [6], magnifying the great need to conduct further research on emphysema, as many are at high risk for developing the disease and its associated complications. With numbers that emphasize the increasingly damaging impacts of emphysema, our work is a critical stepping-stone for future development in therapy and management.

Materials & Methods

Within our research, the methodology was divided into two aspects: synthesis and testing. Before drug synthesis was begun, Tris buffer was prepared by mixing 2.42 grams of Tris and 2.34 grams of sodium chloride in 300 mL of H₂O. Hydrochloric acid was added until the pH was stabilized to 7.21, and H₂O was added until the buffer amounted to 400 mL. L-Alanine- p-nitroanilide (Ala-pNA), a fluorescent reporter group that served to imitate PGP and its function by cutting into PNA, was diluted into multiple concentrations for testing purposes. PGP was not utilized as it was difficult to detect. Instead, Ala-pNA was used because it changes color when hydrolyzed, allowing reliable detection of enzyme activity. Concentrations of 1.0 mM, 2.0 mM, 4.0 mM, 8.0 mM, 12.0 mM, 16.0 mM, 20.0 mM, and 40 mM Ala-PNA concentrations were created by diluting Ala-PNA with water.

Drug synthesis began when 0.103 mL of acetic anhydride, 0.004 mL of pyridine, and 0.1 g of 4-benzylphenol in 5 mL of dichloromethane were mixed together in a bulb flask. 4-benzylphenol was diluted in dichloromethane because the reactant was a solid, and 5 mL of dichloromethane were used since 10 mL of dichloromethane was needed for every millimol of 4-benzylphenol. The compound was placed on a stirring plate overnight for 18 hours to ensure that the reactants were thoroughly mixed. After 18 hours had passed, thin layer chromatography (TLC) was run on the compound. TLC is a method for analyzing mixtures by separating in the mixture, and consists of three steps as follows: spotting, development, and visualization. All of the reactants were diluted with 1:3 ethyl acetate, since pure chemicals produce hazy and unclear dots when run through TLC. Spotters, glass capillary tubes utilized to “spot” the silica gel coated plates, were made by applying heat to glass pipettes and pulling them when warm. Silica gel coated TLC plates were cut into proportionate squares and marked accordingly at the starting and ending points with a pencil. Each of the diluted reactants and product were spotted once on each “lane” of the TLC plate, and immediately placed in a bowl topped with a watch glass with 1:3 ethyl acetate in it. The spotted TLC plate was placed so that the 1:3 ethyl acetate did not pass the marked starting point, as the solvent would instantaneously cause the spots to move along with it. After the 1:3 ethyl acetate traveled up to the ending point, the plate was removed from the bowl and placed under a 234 nm wavelength UV light. By comparing the lanes with diluted reactants to the lane with the compound, we were able to compare the spots with those of the compound, and determine which spot was new and likely represented the product. After clearly identifying the product, the compound was attached to the Buchi Rotovapor® machine to remove the methylene chloride solvent and isolate the product [7].

A separation process was conducted to serve as a purification step that removes the residual impurities was performed utilizing 50 mL of ethyl acetate, 25 mL of H₂O, 25 mL of 10% copper sulfate, and 25 mL of brine. The product was mixed with 50 mL of ethyl acetate in a separatory funnel, and vigorously shaken until the two solutions appeared to be separated. Then, the separatory funnel was turned open so that all of the ethyl acetate phase could be removed from the flask. This process was repeated with the water, copper sulfate, and brine solutions. After the copper sulfate solution was removed from the flask, TLC was conducted on the remaining product ethyl acetate phase to ensure that no pyridine was remaining in the product, as copper sulfate was accountable for removing any excess pyridine. The brine solution ensured that all excess water was removed from the ethyl acetate solvent. Upon the completion of the separation process, the product was transferred to a beaker, where solid sodium sulfate was added to rid the product of any residual water, serving as a drying agent. The solid sodium sulfate was filtered out using a funnel, and the solvent mixture was concentrated using a Buchi Rotovapor® machine to isolate the drug.

Column chromatography, a process used to purify liquids through the separation of the sample by partitioning between the stationary phase silica gel and 1:9 and 1:19 ethyl acetate-hexanes mobile phase, was conducted as the final purification step [8]. Silica gel was packed to the rim of the separating funnel, and 100 mL of hexanes were poured into the funnel. A tube attached to the pressure and air pumps was inserted into the funnel to accelerate the rate at which eluents would flow out of the funnel and into test tubes. After 100 mL of hexanes were filtered out of the funnel until the liquid reached the top of the silica gel, the process was repeated with 1:9 and 1:19 ethyl acetate-hexanes after the product was washed with hexanes and poured into the funnel. Increasing ratios of ethyl acetate to hexanes were used since they would increase the rate of product elution. After all ethyl acetate-hexanes solutions were filtered out, TLC was run on each of the test tubes to identify where the product was located. By matching the lanes that consisted of spots with the corresponding test tubes, the tubes that displayed spots were mixed together in a bulb flask and attached to the Buchi Rotovapor® machine to isolate the drug.

The bulb flask consisting of the drug was washed with 1:3 ethyl acetate-hexanes to ensure that the remaining product on the sides of the flask were included in the drug remaining at the bottom of the flask. The mixture of 1:3 ethyl acetate-hexanes and the drug was pipetted into a clean bulb and attached to the Buchi Rotovapor® machine to remove excess 1:3 ethyl acetate-hexanes solvent. Then, the bulb flask was attached to a Buchi vacuum pump to get any remaining solvent excess out of the product. After the drug was vacuumed for 5-10 minutes, the final product was obtained. In order to ensure that the final product was the desired drug, nuclear magnetic resonance (NMR) spectroscopy was run through the Bruker 400 megahertz NMR on the drug to determine the structure of the organic compound. Gas chromatography-mass spectrometry (GC-MS) analysis was run on the drug to identify substances within the drug sample, and also to identify the chemical formula of the drug. Both of these analyses confirmed the purity of the drug, and ensured that the acquired product was the desired drug- 4-AcDM.

During the testing stage, a BioTek microplate reader was utilized to run enzyme assays. Using a 96-well plate, each well consisted of 50 microL of LTA4H enzyme solution, 50 microL of Tris buffer, 50 microL of drug solution, and 50 microL of Ala-pNA solution. Each row consisted of a different drug concentration, starting from 0 mM, 1 mM, 5 mM, 10 mM, 20 mM, 40 mM, 80 mM, and ending with 160 mM. Various drug concentrations were created by diluting the drug with 20% DMSO buffer. Every three columns consisted of a different Ala-pNA concentration: one plate held 12 mM, 16 mM, 20 mM, and 40 mM of Ala-pNA, while another held 1 mM, 2 mM, 4 mM, and 8 mM of Ala-pNA. The reporter groups were inserted last to ensure that the reaction would occur in the microplate reader when the plate was read. After a plate was read and optical density values were produced, data was interpreted accordingly by utilizing the GraphPad Prism 5 application.

The lab director trained the student researchers in running instrumentation in the lab and supervised the research. All procedures for this project were conducted and carried out by the student researchers.

Results

When the 96-well plate consisting of enzyme, drug, buffer, and reporter group was placed into the BioTek Plate Reader, each well produced an optical density value that represented the absorption by the sample. For each drug concentration, graphs comparing varying Ala-pNA concentrations (as the x values) and optical densities (as the y values) were initially created, and the linear portions of each graph were isolated to determine the graphs’ equations. With the linear formula, the y value represented optical density, x represented Ala-pNA concentration, and m and b both served as constants. To convert the optical density values to Ala-pNA concentrations instead, the formula, y = 1575 x + 0.9177, was rearranged to x = (y - 0.9177) / (1575). This formula was found by isolating the graph of Ala-PNA concentration vs. averages of optical densities to ensure that the formula was a display of collective data, rather than solely one drug concentration. Using this formula, the optical density values were converted to PNA concentration values.

After attaining PNA concentration values, the GraphPad Prism application was utilized to construct graphs for each drug concentration that displayed the trend between time and PNA concentrations. Each kinetic trend represented time in minutes, and PNA concentration values were obtained from the values that were calculated using the initial formula. For each of the graphs, the initial velocities, V0, were calculated by finding the slope of the graph from 0 to 5 minutes. To construct the final Michaelis Menten plot, which displays the effect of Ala-pNA concentrations on the initial velocities for each concentration of drug, the lower Ala-pNA concentrations (0.00 mM, 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM) were placed on the x-axis, while the initial velocities found by calculating slopes of the individual time vs. PNA concentration graphs were placed on the y-axis. Each color represented a different drug concentration, while the ranges for the x and y axis were preset based upon the input Ala-PNA concentration and initial velocity values.

The resulting data, in the form of a Michaelis-Menten plot, led to the conclusion that with a greater drug concentration, a greater initial velocity of the reaction resulted. Additionally, with an increasing Ala-pNA concentration, more pNA resulted, indicating that the greater the presence of Ala-pNA, the greater the velocity of cutting. Hence, if a drug produces more pNA, increasing reaction velocities result.

Next, several enzyme kinetics tests were run. Vmax (mol/sec), the maximum rate of reaction, increased with increasing drug concentrations. Since Vmax is under the conditions of a sufficient amount of substrate molecules to saturate the enzyme’s active sites, the data confirmed that the 160.0 mM drug accelerated the rate at which the enzyme catalyzed the reaction. Km (mol/L), the concentration of substrate needed to achieve half of the Vmax , was greatest at the drug concentration of 160.0 mM. The larger Km value, 0.08869 mol/L, meant that great amounts of substrate are necessary to saturate the enzyme, also indicating that the enzyme has a low affinity for the Ala-pNA reporter group and is less specific. Kcat (1/sec), the turnover number for the AP activity, was greatest at the drug concentration of 160.0 mM. As Kcat represents the rate of the enzyme, the value stands for how fast the enzyme “makes” pNA from Ala-pNA, and is expressed per unit time.  The large Kcat value means that with 160.0 mM of drug, the enzyme produces pNA at the fastest rate from Ala-pNA. Kcat/Km , the catalytic efficiency, was the lowest at 160.0 mM of drug. The Kcat/Km value represents the number of times the substrate is converted to product per unit of time. To place the interpretation of Kcat/Km into context, a “perfect” enzyme would have a very high Kcat (high speed) but a low Km (specific). However, in reality, reducing the Km will slow the Kcat , because the substrate is bound tightly to the enzyme and slow down the turnover number. Although we look at Kcat / Km to determine a maximum number, our drug showed a low Kcat/Km value since a higher value exists with drugs that block enzyme activity (involving EH activity); our was contrary and activated enzyme activity, leading to a lower value with an effective drug.

The initial research question asked was: how do we synthesize a compound that will target the aminopeptidase pathway of the LTA4H enzyme? The results presented address this question by proving the efficacy of our compound, 4-AcDM. The rate at which 4-AcDM cuts Ala-pNA increases with increasing concentrations of the drug, confirming that the compound targets the AP pathway of the enzyme in a quicker and more thorough manner. Within the graphs, we recognized that the highest V 0 curve was found representing the highest drug concentration of 160.0 mM, and diminished as drug concentrations lessened. In addition, enzyme kinetics prove that our drug accelerated the rate at which the substrate binds to LTA4H. Since the hydrolysis of Ala-pNA  to p-NA mimics that of the hydrolysis of PGP, the drug has potential to lower the progression of EM and resolve PMN activity.

Illustrations

Discussion

The Michaelis-Menton plot obtained showed that with increasing concentration of our drug, 4AcDM, the rate of pNA cutting (and therefore, PGP cutting) is accelerated. Further, the maximum rate of reaction increased with increasing presence of 4AcDM. These enzyme kinetics tests supported the conclusion that the drug was efficient in PGP hydrolysis.

The molecule synthesized in our experiments, 4AcDM, was efficient and activated the AP activity of the LTA4H enzyme. Our work sets itself apart from other emphysema-related treatments because it targets the anti-inflammatory pathway of the disease itself and serves to be an activator of a pathway, rather than an inhibitor. Since most work that are centralized around developing enzyme inhibitors for the EH pathways have proved to be inefficient when tested, enzyme activators for the AP activity were developed instead. In one study conducted by Konstan et al., the effects of LTB4 receptor inhibition on the reduction of airway inflammation proved to be trivial as an increase in the “risk of infection-related adverse events” was possible [11]. Although the inflammatory response could have been suppressed, unexpected infections could have occurred in conjunction, making the lessened inflammatory response insignificant. In another study conducted by Roberts et al., the effects of inhibiting leukotriene (LT) biosynthesis on inducing remission in patient with ulcerative colitis were observed [12]. Although the 5-lipoxygenase inhibitor inhibited LT biosynthesis, the inhibition did not greatly differ from the use of placebo in clinical efficacy. It was Paige et al. who first studied the role of the the LTA4H aminopeptidase in the pathogenesis of emphysema, finding that the AP activity of the enzyme breaks down and clears PGP [4]. Our observations support those of Oliveira et al. who studied the anti-inflammatory pathway. In their experiments, Oliveira et al. observed the effects of the compound known as 4-methoxydiphenylmethane (4-MDM) [5]. Although this compound does activate the AP activity of the LTA4H enzyme, the compound itself is chemically unsound. As Lai et al noted, several properties of 4-MDM make it an undesirable drug treatment for emphysema, including its poor water solubility and an unstable central oxygen atom [9].

However, our work extends and enhances these findings because we have synthesized a novel compound. In our drug, 4AcDM, we replaced the central oxygen atom with a stable carbon atom and attached an acetyl group to increase the overall stability of the compound. The drug, with its enhanced physicochemical properties, is effective in enacting the AP pathway of LTA4H. Enzyme assays confirm that we have synthesized an improved compound that promotes anti-inflammation, extending and improving on Oliveira’s work. With more oxygen atoms and an acetyl group, our drug proved to more efficient at the enzyme level. Further, with increased concentrations of 4AcDM, the rate of AP activity increased, confirming the efficacy of our drug as treatment for the inflammation associated with emphysema.

The final product developed was C15H14O2, comprised of 4-benzylphenol in dichloromethane solvent, pyridine as the catalyst, and acetic anhydride. Our drug is a significant step towards novel emphysema treatment once tested within in-vitro and in-vivo studies because it is efficient at clearing inflammation, ultimately preserving the protective properties of 4-MDM while existing as a more stable compound.

Conclusion

Our research concluded that 4AcDM was efficient when administered in 160.0 mM, as the greatest initial velocity (V 0 ) at which the reaction occurred resulted when the drug was tested upon enzyme assays. Our work addressed the initial research question by synthesizing a compound that was improved through the addition of a carbon atom and acetyl group, which succeeded in producing a well functioning LTA4H enzyme activator. Our results are fully supported by the results described in the report, as other research focused upon the enzyme present the results found in in vivo and in vitro studies. Since our research looked at the drug’s effects in the enzyme level, our results were strongly supported by enzyme kinetics concepts versus those of others’ research.

Experiments performed in the future should work towards improving the water solubility of the drug, and determine the best formulation for in vivo and in vitro studies. If we had more time, we would look into both water solubility and in vivo/in vitro studies, while also looking towards utilizing methyl chloroformate and ethyl chloroformate in place of acetic anhydride. We believe that the addition of a methyl group or ethyl group could have potential benefits, and since both were tested in the preliminary stages of our experiment, reasons behind why the chloroformate solutions failed to react with other reactants would need to be investigated. If we were to start the work today, we would work towards improving the percent yield by being more attentive to amounts of reactants mixed to ensure that the greatest amount of product can be collected at the end. We would run more enzyme assays to validify our results, and run more TLC to assure that the final product is purified to its maximum potential. During column chromatography, varying solutions of ethyl acetate hexanes could be used, rather than simply 1:9 or 1:19. Using more solutions of ethyl acetate hexanes would allow more of the product to come out at faster rates. Questions that still remain to be answered include, what other reporter groups, other than Ala-pNA, could be used to portray the activity of PGP in a more noticeable and researchable manner? What other roles could the LTA4H enzyme have other than its function in lung diseases? Could it have a significant role in common diseases such as cancer, and could our drug have multiple uses? How could we develop our drug into one that is in crystallized form, for use especially in clinical trials?

References

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