Clinical Test for a New Drug for Treating Arteriosclerosis - Assignment Example

Clinical Test for a New Drug for Treating Arteriosclerosis Clinical Test for a New Drug for Treating Arteriosclerosis Atherosclerosis Atherosclerosis, also known as the hardening of arteries, is a slowly occurring disease characterized by the clogging and hardening of arteries thus leading to vascular dementia, heart attacks, and strokes. Atherosclerosis results from the accumulation of a combination of cholesterol, calcium, fat and other substances into plaques within the arteries. Once the plaque hardens, the passage of blood in the arteries narrows hence hardening and loss of flexibility of the arteries. If the plaque does not harden, it breaks free from the arterial walls and forms a blood clot which has the capability of blocking blood flow to an individual’s vital organs hence death. The magnitude and extent of the effects of atherosclerosis vary in accordance to the arteries that have been narrowed and clogged with plaque. For instance, peripheral artery disease is a result of the clogging and narrowing of the arteries supplying the oxygenated blood to the arms and legs. Cerebrovascular disease, stroke and Transient Ischemic Attack (TIA) where an individual loses brain function but recovers completely after a day result from clogging and narrowing of the arteries supplying blood to the brain. Coronary artery disease is a result of the narrowing of the arteries of the heart. Symptoms and Risk Factors Similarly, the symptoms of atherosclerosis vary according to the artery that has been affected. However, according to the University of Maryland Medical Center (2013, p.1), most symptoms only manifest when the affected artery is 40 percent clogged with plague. Common symptoms include body weakness, pains in the chest, abdomen, neck, back and arms, shortness of breath, muscle weakness, dizziness, and hair loss. These symptoms manifest as different diseases depending on the affected arteries as explained earlier. Nonetheless, the exact cause of atherosclerosis is unknown as most studies point to factors that increase the risk such as smoking, diabetes, lack of physical exercise, unhealthy diet, and obesity. Intervention According to Zadelaar et al (2007, p.1706), atherosclerosis is still the main cause of “morbidity and mortality in people living in industrialized societies despite the tremendous advancement its biological understanding, and significant advancement in treatment methods.” The high mortality rates resulting from atherosclerosis can be attributed to some dominant risk factors such as low-density lipoprotein (LDL) cholesterol and very low-density lipoprotein (VLDL) cholesterol also referred to as hypercholesterolemia. In order to reduce the cases of atherosclerosis, scientists have been focusing, for the past twenty years, on means by which hypercholesterolemia can be treated (Zadelaar et al., 2007, p.1706). These studies have resulted in the development of cholesterol-lowering drugs, especially of the statin class, as the most feasible and highly potent hypercholesterolemia treatment strategies. The success rate of the statin class of drugs in reducing deaths due to cardiovascular complications by lowering cholesterol levels is very high (Kostner and Cauza, 2005, p. 1). However, more than 60 percent of patients under statin-based treatment eventually experience adverse coronary episodes (Kostner and Cauza, 2005, p. 1). Zadelaar et al., (2007, p.1706) also state that due to the overdependence of statin-based drugs in the treatment of atherosclerosis, recent studies have focused on the development of new therapeutics that can either be used exclusively or in combination with a statin in targeting more than one risk factor. These new drugs are designed to target risk factors such as hypertension, hypertriglyceridemia, type-2 diabetes, low high-density lipoprotein (HDL), and insulin resistance on top of hypercholesterolemia as identified by classical epidemiology (Zadelaar et al., 2007, p.1706). Bursill, Channon and Greaves, 2004, p. 145 claim that the progression of atherosclerosis requires chronic systemic and vascular inflammation as they contribute to atherogenesis. However, due to the complexity and chronicity of the process of the development of atherosclerosis, the atherogenic processes and mechanisms in human beings is very challenging to define. Moreover, despite the rapid progress in noninvasive disease detection procedures due to technology advancement, sequentially characterize lesions in an individual poses a significant challenge. Due to the aforementioned reasons, this study description of a test for a new drug to treat atherosclerosis mainly relies on animal models in the verge of understanding the causal factors and pathogenic steps followed by the disease. Based on several factors, mice are the best animals to use in testing the new drug to treat atherosclerosis. For instance, the small size and high adaptability to new surroundings makes mice easy to maintain and house. Additionally, the gestation period of mice is short hence they reproduce very quickly. Considering the study of a new drug takes several years, mice are also the most viable animals as they have a short lifespan hence intergenerational changes can be monitored in relatively short periods of time. A more critical reason for using mice is because “they closely resemble human beings genetically, biologically and behaviorally hence most human disease symptoms can be replicated in mice” (Melina, 2010, p.1). The responses of mice to a new drug meant to target the different risk factors for atherosclerosis such as inflammation, hypercholesterolemia, hypertension and hypertriglyceridemia dominate this document. Study Design For the testing of this new drug, this study will adopt the Randomized Controlled Trial (RCT) because it is among the most common designs for experimental medical studies especially in testing new drugs and therapeutics. Using this design, the candidates for the study are allocated by chance using methods such a tossing a coin creating study groups with equivalent factors such as age, sex, and reactions to medication. This design’s upper hand is the fact that it avoids confounding that is confusing the effects of variables. Such variables might include the new drug and other factors such as age and sex which might differ in the different study groups. In order to ensure that the differences in the study’s outcomes are results of the new experimental drug and not the result of other factors, this study design ensures that all study groups are comparable by all means. In some instances, some confounding factors are known hence the researcher could match the study groups based on the factor for example balancing the number of male mice and female mice. However, even in such an instance, only the effects of the known confounding factor can be avoided. For instance, an unknown biological limitation could modify the expected action of the drugs. Using Randomized Controlled Trial, the results of the study are protected from all confounding factors whether known or unknown hence the measuring and controlling of each factor individually is unnecessary. A study of this dimension is bound to involve study groups that are not absolutely and perfectly identical despite being selected randomly. To deal with this, statistical tests will be employed in the verge of determining whether the expected difference observed in the experiment’s outcome is a reflection of natural variability due to chance or a representation of a significant statistical difference. In order to determine the strength of the correlation between the new treatment and the reduction of risk factors for atherosclerosis, a rank correlation will be used. The animal models will be arranged in classes according to their weight change, cholesterol level disparity before and after using the drug and general activity levels on separate occasions. This will then be combined with a chi-square test to indicate the association between the effects of the new treatment and the reduction of atherosclerosis risk factors by comparing the frequency distribution observed in the mice models with expected results under a null hypothesis. In order to determine that the difference before the intake of the drug and after is significant, an analysis of variance (ANOVA) will be employed. This is because ANOVA gives a comparison within each sample study group and between each sample study group. This data will indicate the most appropriate dosages for the drug and suggest the most feasible intake method for the drug. Probable Mouse Models for Testing the New Drug Due to low levels of proatherogenic LDL and VLDL, and high levels of antiatherosclerotic HDL, wild-type mice have a higher resistance to atherosclerosis. Therefore, all mouse models for atherosclerosis are based on “perturbations of lipoprotein metabolism which is achieved through strategies such as dietary and genetic manipulations” (Lensik, Haskell and Charo (2003, p. 333). There are several common mice models to select from in the clinical trial and biochemical arenas. These models include the most common apolipoprotein E-deficient mice (ApoE-/- mice). In this mouse model, the apoE gene is deleted leading to severe hypercholesterolemia which then culminates in spontaneous atherosclerosis. The liver and macrophages synthesize ApoE, which has significant antiatherogenic properties and functions. ApoE serves as a ligand for the cell surface lipoprotein receptors such as the LDLr-related proteins and the LDL-receptor (LDLr) as it is a constituent of plasma lipoproteins. Therefore, ApoE promotes the uptake of atherogenic compounds from the circulatory system. The homozygous deletion of the ApoE gene in the ApoE-/- mice results in the failure of LDLr- and LRP-mediated clearance of plasmic lipoproteins such as LDL and VLDL which then culminates in an increase in their levels. Consequently, ApoE-/- mice develop spontaneous atherosclerosis lesions even when feeding on a standard cholesterol-free chow diet with a fat content less than 40g/kg. The atherosclerotic lesions developed in ApoE-/- mice resemble those developed in humans and these mice also rapidly develop atherosclerosis hence this model is widely used. However, the absence of the ApoE gene in ApoE-/- mice amplifies the plasma cholesterol levels of these mice even when fed on low-fat and cholesterol-free diets. Moreover, the high plasma cholesterol levels in ApoE-/- mice are limited to particles of VLDL and not of LDL as in human plasma. The ApoE protein is also involved in foam cell formation and reverses cholesterol transport hence its absence in ApoE-/- mice makes it challenging to study the aforementioned processes and the effects of the drugs with respect to them. There is also the LDL receptor-deficient mice model (LDLr-/- mice). In this mouse model, atherosclerosis results from feeding the mice with a lipid-rich diet. The mutations of the gene for the LDL receptors (LDLr) results in familial hypercholesterolemia in humans in the same way as LDLr-/- mice show elevated plasma cholesterol due to its lack thereof. This model develops atherosclerosis gradually unless feeding on a high-fat, high cholesterol (HFC) diet. However, the most significant advantage of this model is that unlike ApoE-/- mice, the lipoprotein profile of LDLr-/- mice resembles that of humans as it is dominated by LDL particles. However, the most recent emerging model, the ApoE*3Leiden (E3L) transgenic mouse, has more advantages than LDLr-/- mice and ApoE-/- mice especially when testing a new drug to treat atherosclerosis. In this mouse model, a mutated form of the human apoE3 gene is introduced making the E3L mice phenotypically hyperlipidemic hence developing atherosclerosis after feeding on cholesterol. When there is a mutation in the APOE3 gene in humans, the result is a rare and dominant-negative mutation called the ApoE*3-Leiden. In order to generate an ApoE*3-Leiden (E3L) transgenic mouse, a C57BI/6 mouse is injected with the human APOE*3-Lieden gene construct which also bears the APOCI gene and the element that boosts the expression of the APOE and APOCI genes. E3L mice express the ApoE protein even though the process by which these mice clear ApoE-containing lipoproteins is less dramatic compared to ApoE-/- mice. When feeding on a regular chow diet, E3L mice show significant increases in levels of plasmic cholesterol and triglycerides. Unlike wild-type mice, E3L mice are, additionally, highly responsive to cholesterol-, sugar-, and fat-containing diets which results in strong elevations in their plasmic cholesterol and triglyceride levels. The elevated levels are dominated by a significant increase in LDL- and VLDL-sized lipoprotein particles. E3L mice are also suitable for testing a new drug since plasma lipid levels can be easily monitored and adjusted to desired concentrations by titrating the intended amount of cholesterol and sugar in the mice’s diet. Moreover, E3L mice’s cholesterol levels do not exceed 25mmol/L even on a high-fat, high cholesterol (HFC) diet hence they represent a moderate model for hyperlipidemia. The main advantage of E3L mice over other mice models discussed in the document is that the E3l mice are more sensitive to lipid-lowering drugs. Since levels of plasma cholesterol and triglycerides respond strongly to changes in the production of hepatic VLDL, any drug that influences the production of chylomicron and VLDL culminates in parallel effects in the mice’s plasma cholesterol and triglyceride levels. This makes E3L mice more sensitive and responsive to hypolipidemic compounds with cholesterol-lowering properties compared to LDLr-/- mice and ApoE-/- mice. Moreover, the development of lesions in E3L mice has all the characteristics of human vascular pathology. The development of lesions in E3L mice, just as in humans, is time-dependent and progresses from fatty streaks to mild, moderate, and severe plaques. This makes this model the most suitable for testing a new drug to treat atherosclerosis. Materials and Methods The first step will obviously be the recruitment of patients with atherosclerosis. The best channel to do this would be to patients who were referred to a medical research institution for a carotid endarterectomy (CEA). Once patient have been recruited, the will be studied consecutively after they have been fully informed about the research. Appropriate ethical and consent-seeking channels should be followed and the study has to be approved by relevant institutions and authorities. Once patients have been recruited, venous blood samples will be collected from the cubital vein and centrifuged after clot formation to obtain samples of the serum. Cross-sectional immunohistological analysis for the carotid tissue specimens ought to be performed, and all atherosclerotic lesions ought to be quantified in accordance with the guidelines provided by the British Heart Foundation. Using a controlled heat and pH 6.0 citrate buffer, the researcher will perform an antigen retrieval procedure. Atherosclerosis is mainly characterized by the accumulation of macrophages which are laden with lipids in the subendothelial area of arteries. Recent studies indicate that chemokines facilitate the migration of monocytes from the blood to the circulatory vessel walls. Fractalkine is one such chemokine. Therefore, anti-human antibodies will be used to target fractalkine, macrophages, endothelial cells, vascular smooth muscle cells and CX3CR1. Using the APAAP ChemMate Detection Kit, primary antibodies will be detected. The samples will be blocked with normal goat IgG (Invitrogen) for immunofluorescent staining. The blocking will be done for half an hour after which the samples will be labeled at 4 degrees Celsius with antibodies targeting the human CX3CR1 and fractalkine. The antibodies will be diluted in PBS with a composition of 0.3 percent Triton X-100 and 0.5 percent BSA. Once samples are washed and incubated, they were prepared for mounting and viewing under an electron microscope. Suitable negative control for this procedure would be rat IgG2b and rabbit IgG. This new drug is aimed at targeting risk factors other than hypercholesterolemia by being administered in combination with other clinically tested and approved statins. However, the primary function of the drug is to promote cholesterol secretion and prevent excessive absorption of cholesterol from food. To be particular, the medicine is designed to inhibit selectively the uptake and absorption of cholesterol by the enterocytes of the ileum. By inhibiting the intestinal uptake of both dietary and biliary cholesterol, the drug is intended to reduce the susceptibility to atherosclerosis by 97 percent. ApoE*3Leiden (E3L) transgenic mice will be generated by introducing a mutated form of the human apoE3 gene (APOE*3-Lieden gene construct) to C57BI/6 mice. The resulting mice will then be used for this study. The mice will be fed on a normal rodent chow diet with 4.5 percent fat content for fourteen days. After the end of this period, the mice will be weighed before being introduced to a high-fat Western diet with 0.15 percent w/w cholesterol content and 21 percent anhydrous milk fat. The mice will split into different groups with one being fed on the Western diet for 5 weeks, the other for 10 weeks and the other for 15 weeks. After every group has terminated its allocated period of feeding on the high-fat diet, the individual members of the group will be weighed again. From every group in the previous stage of the study, some mice will be sacrificed in order to assess the extent to which the atherosclerotic lesions have developed in the proximal aorta of the mice from different groups. To determine the quantity of the cellular content of the atherosclerotic lesions, the sections being studied will be stained with antibodies targeting macrophages, smooth muscle cells, and endothelial cells. The sections under study will be air dried for about an hour then fixed in formalin (10 percent) for half an hour at room temperature. Once the atherosclerotic lesions have been quantified, the remaining rats will be divided equally in their groups, and each sub-group will be administered with known and approved standard statin treatment or the new drug only. The group under the standard treatment will act as a control as it will provide a baseline for the comparison of the efficacy of the new drug working on its own. After 5 weeks, 10 weeks and 15 weeks (according to the groups), some mice from each sub-group will be sacrificed as the extent to which the atherosclerotic lesions have developed in the proximal aorta of the mice will be quantified again using the same method. This will indicate the effectiveness of both the standard treatment and the new drug. The mice will then be weighed again then a combination of both the new drug and the standard treatment administered. This step will provide information on whether the new drug has optimal efficacy on its own or when combined with other statins. This step will also highlight whether the standard treatment is more effective than the new drug when working solely or when working in combination with another drug. After similar periods, the mice will then be weighed, and the extent of atherosclerotic lesions quantified again. This will be the final determinant of whether the new drug effectively reduces fractalkine thus inhibiting atherogenesis. References Bursill, C., Channon, K., & GREAVES, D. (2004). The Role of Chemokines in Atherosclerosis: Recent Evidence from Experimental Models and Population Genetics. Curr Opin Lipidol, PubMed. US National Library of Medicine. National Institute of Health. Accessed 10th June 2015 from: http://www.ncbi.nlm.nih.gov/pubmed/15017357 Canada University. (2012). Medical Study Designs. Society, the Individual, and Medicine. Accessedon 10th June 2015 from: http://www.med.uottawa.ca/SIM/data/Study_Designs_e.htm Charo, I., & Taub, R. (2011). Anti-inflammatory Therapeutics for Treatment of Atherosclerosis. National Rev Drug Discovery. Vol 10, No. (5), (p.365-376). Accessed 10th June 2015 from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947588/ Gregg, B., Lumeng, C., & Bernal-Mizrachi, E. (2014). Fractalkine Signaling in Regulation of Insulin Secretion. Mechanisms and Potential Therapeutic Implications? Islets, CrossMark. Vol 6, No. 1 (1-6). Accessed 10th June 2015 from: http://www.tandfonline.com/doi/pdf/10.4161/isl.27861 Hafiane, A., & Genest, J. (2013). HDL, Atherosclerosis, and Emerging Therapies. Review Article, Hindawi Publishing Corporation. Vol 2013, ID. 891403. (p.1-18). Accessed 10th June 2015 from: http://www.hindawi.com/journals/cholesterol/2013/891403/ Jellinger, P., Mehta, A., Handelsman, Y., & Sheperd, M. (2012). America Association of Clinical Endocrinologists’ Guidelines for Management of Dyslipidemia and Prevention of Atherosclerosis. AACE Guidelines. Endocrine Practice, Vol 18, No. 1, (p.1-78). Accessed 10th June 2015 from: https://www.aace.com/files/lipid-guidelines.pdf Kam, K. (2014). Breakthroughs in Atherosclerosis Treatment. New Research May Lead to New Drugs for Heart Disease. Heart Disease Health Center. WebMD the Magazine. Accessed 10th June 2015 from: http://www.webmd.com/heart-disease/features/breakthroughs-in-atherosclerosis-treatment Kostner, K., & Cauza, E. (2005). HDL-Therapy: The Next Big Step in the Treatment of Atherosclerosis? Future Medicine, Future Cardial, Vol 1, No. 6, (p.1-7). Accessed 10th June 2015 from: http://www.researchgate.net/profile/Karam_Kostner/publication/26870261_HDL_therapy_the_next_big_step_in_the_treatment_of_atherosclerosis/links/02e7e524c823a4297f000000.pdf Lensik, P., Haskell, C., & Charo, I. (2002). Decreased Atherosclerosis in CX3CR1-/- mice Reveals a Role for Fractalkine in Atherogenesis. The Journal of Clinical Investigation. Vol 111, No. 3. (333-340). Accessed 10th June 2015 from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC151849/pdf/JCI0315555.pdf Melina, R. (2010). Why Do Medical Researchers Use Mice? Live Science. Accessed 10th June 2015 from: http://www.livescience.com/32860-why-do-medical-researchers-use-mice.html Orekhove, A. (2013). Anti-atherosclerotic Drugs from Natural Products. Natural Products Chemistry and Research. Vol 1, No.4 (p.1-6). Accessed 10th June 2015 from: http://esciencecentral.org/journals/antiatherosclerotic-drugs-from-natural-products-2329-6836.1000121.pdf Stolla, M., Pelisek, J., Von Bru¨HL M-L., Scha¨Fer, A., Barocke, V., ET AL. (2012). Fractalkine Is Expressed in Early and Advanced Atherosclerotic Lesions and Supports Monocyte Recruitment via CX3CR1. PLoS ONE Vol 7, No. 8: e43572. doi:10.1371/journal.pone.0043572 (p.1-9). Accessed 10th June 2015 from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423360/pdf/pone.0043572.pdf The Jackson Laboratory. (2014). Advantages of the Mouse as a Model Organism. Leading the Search for Tomorrow’s Curves. Accessed 10th June 2015 from: http://research.jax.org/mousegenetics/advantages/advantages-of-mouse.html University Of Maryland. (2014). Atherosclerosis. University of Maryland Medical Center. Accessed 10th June 2015 from: http://umm.edu/health/medical/altmed/condition/atherosclerosis Zadelaar, S., Kleemann, R., Verschuren, L., Hoorn, J., Princen, H., & Kooistra, T. (2007). Mouse Models for Atherosclerosis and Pharmaceutical Modifiers. Arteriosclerosis, Thrombosis and Vascular Biology. Journal of America Heart Association. Vol 27, No. 1 (1706-1721). Accessed 10th June 2015 from: http://atvb.ahajournals.org/content/27/8/1706.full.pdf Read More