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Breast cancer occurs when there is an uncontrolled growth of mutated cells from breast tissues. A cluster of cancerous cells or primary tumor will result from the excessive proliferation of mutated cells. Cancer cells will produce a growth factor, vascular endothelial growth factor (VEGF) which will stimulate angiogenesis Angiogenesis, the process of formation of new capillary system, will connect existing blood vessels to the tumor allow it to grow in size beyond 1-2mm as the tumor can obtain sufficient nutrients and oxygen. This will enable tumor cells to enter circulation and metastasis may occur when cancerous cells leave the primary tumor and spread to other parts of the body via blood vessels or lymphatic system. This may result in secondary tumors which may form at distant sites. A malignant tumor is a tumor that is spread by metastasis and breast cancer refers to a malignant tumor that has developed from cells in the breast.
There are mainly 2 forms of breast cancer: pre-invasive and invasive. In pre-invasive breast cancer, cancer cells cannot spread and are confined to the breast s ducts or lobules whereas in invasive breast cancer like IDC (Invasive/Infiltrating Ductal Carcinoma) which is the most common type of breast cancer, the invasive ductal carcinoma can spread to the lymph nodes and may even spread to other parts of the body eventually. 
The cause of breast cancer in some cases may be due to genetic disposition due to the inheritance of oncogenes which are mutated forms of proto-oncogenes. Proto-oncogenes are genes whose normal products stimulate cell division. However a gain of function mutations in the proto-oncogenes will convert the proto-oncogenes to oncogenes which are cancer causing. An increase in the amount of proto-oncogene s protein product or an increase in the intrinsic activity of each protein molecule will result from an oncogene that results from a genetic change.
BRCA1 and BRCA2 are tumor suppressor genes which produce a protein: breast cancer type 1 susceptibility protein and breast cancer type 2 susceptibility protein respectively. Mutations of BRCA1 and BRCA2 are linked to inherited breast cancer. Inheriting a BRCA1 or BRCA2 will increase the risk of getting breast cancer.  LCIS or lobular carcinoma in situ signifies that a women is at considerably higher risk than normal of developing a breast cancer in future. 
In our report, we will be focusing on the action of Raloxifene on breast cancer prevention in postmenopausal women.
2. Drug Discovery Process
Raloxifene, also known as Evista is one of the products marketed by Eli Lilly and Company. It can act as both agonist (on bone and lipid metabolism) and antagonist (on uterine endometrium and breast tissue). Currently, it is being used to treat and prevent osteoporosis and invasive breast cancer in postmenopausal women. 
Figure 1: Structure of raloxifene 
The discovery of Raloxifene was an effort to prepare nonsteroidal antiestrogens that act as better antagonist for estrogen receptor and less intrinsic estrogenicity than those that were present (eg. tamoxifen).
Figure 2: Synthesis of Raloxifene 
Structure Activity Relationship was carried out by synthesizing and testing a series of 3-aroyl-2-arylbenzo[b] thiophene derivatives.  MCF-7 human mammary tumour cell line was used for in vitro test for determining the anti-proliferative activity of the derivatives. It proved to be effective in inhibition of the oestrogen-dependent proliferation.  As shown, raloxifene has IC50 value of 0.2nM, which is better as compared to tamoxifen (IC50= 200nM) . In vivo animal tests were then carried out. Rat uterotropic/ antiuterotropic bioassay (in vivo)  was used. Monkey and rabbit tests were also carried out to test the effect of raloxifene on chloresterol levels. [36, 37] The pharmacokinetic profile of the drug was then optimized based on the results obtained. Animal test of the compounds showed that the compound Raloxifene had high degree of inhibition of estrogen receptor, Kd ~50pmol/l (even higher than tamoxifen Kd ~2nmol/l)  as shown in Figure 1 and greater binding affinity than estradiol as shown in Figure 2. 
Figure 1: Uterotropic and Antiuterotropic activity of 3-Acyl-2-arylbenzo[b]thiophene derivatives in immature rats 
Figure 2: Rat uterine cytosol estrogen receptor relative binding affinity of 38 and 44 (estradiol = 1.0). 
The major metabolites of raloxifene were then used to test the efficacy in vitro and in vivo. The conjugates have considerably less affinity for estrogen receptor and have less potency in inhibiting cell proliferation than raloxifene. This confirmed that the pharmacological effect is due to raloxifene and not its conjugates. 
Preclinical trials and clinical trials were then carried out to get in vivo data to support the usage of raloxifene in reduction in risk of breast cancer, see Figure 3 and Figure 4. 
Figure 3: Effect of control, raloxifene, 9-cis-retinoic acid and their combination on tumour number 
Figure 4: Kaplan-Meier analyses of percentage of disease-free patients after treatment with raloxifene and placebo.
After the toxicological and safety tests and approval by FDA in 1997 , the drug is then mass produced and marketed.
It is first acknowledged as an effective drug in reducing risk of breast cancer in postmenopausal women by National Cancer Institute in 2006 and its usage on reducing breast cancer in postmenopausal women with osteoporosis or high risk for invasive breast cancer was later approved in 2007 by U.S. Food and Drug Administration.
3. Mechanism of action (Pharmacodynamics)
a) Brief definition of intracellular receptor
Raloxifene is an antagonist which targets the intracellular receptor (oestrogen receptor), in which its specific mechanism of action will be explored further below. 
The structure of intracellular receptor is as follows:
Figure 6: Intracellular receptor
(Diagram adapted from An Introduction to Medicinal Chemistry by Gary L. Miessler and Donald A. Tarr, Pearson)
The ligand first crosses the cell membrane into the cell and binds to the steroid binding region of the intracellular receptor, which is near the C terminal. The binding of the receptor causes a conformational change in the receptor, leading to dimerisation. The dimerisation causes another conformational change in the receptors, opening up a binding region for co-activator protein to bind, forming a transcription complex. The complex can then bind to DNA via the DNA binding region with zinc fingers . This can either activate or inhibit protein synthesis by switching protein synthesis on or off.
Figure 7: Mechanism of ligand binding to intracellular receptor
(Diagram adapted from An Introduction to Medicinal Chemistry by Gary L. Miessler and Donald A. Tarr, Pearson)
b) Structure of estrogen receptor
It has a variable N-terminus, containing the AF-1 domain hormone-independent activation through coactivator binding and transcriptional activation of target genes; DNA binding region which is near to the center, binds to specific gene sequences and contains 2 zinc fingers that also aid in the dimerization of the receptor; ligand binding domain, including the AF-2 domain that interacts with ligands as well as binds to coactivators and corepressors. 
c) Normal effect of estrogen on estrogen receptor
The estrogen first binds to the ligand binding domain and causes conformational change to the estrogen receptor, forming a complex which can then bind to the estrogen response elements. It can then binds to coactivators and corepressors, which control the transcription activity and affect gene expression by increasing and reducing transcription. 
Figure 8: Estrogen hormone (H) binds to the estrogen receptor at ligand binding domain (LBD) causing conformational change of ER and dimerisation and subsequent binding to the estrogen response element (ERE) upstream of the gene to be transcribed. It can recruit coactivators and corepressors through mainly AF2. The result is the activation of transcription complex and stimulation of cell proliferation.
There are multiple forms of estrogen receptors, mostly ERa and ERb, which are not isoforms of each other because they are different proteins encoded by different genes. Their distributions in the various tissues differ and they have different activity profile.  Both of them can form heterodimer and result in conformational change, allowing various proteins (eg. coactivator) to bind to form the coactivator complex. 
One example of the coactivators that interact with estrogen receptor is histone acetyltransferase, which catalyzes the transfer of acetyl groups from acetyl CoA to lysine residues in N-terminal of histone proteins, changing the positively charged lysine to uncharged lysine [40, 44]. This results in 1) a reduction of affinity of histone complex for DNA, exposing DNA regions for transcription proteins to bind ; 2) recruitment of transcription factors by interaction between acetylated lysine residues and bromodomain ; 3) initiation of active remodelling of the chromatin structure for the DNA binding proteins to gain access to their attachment sites . After binding with the coactivator, the complex can then enhance transcription initiation by helping RNA polymerase to achieve promoter clearance or control other substeps of transcription (eg. elongation, RNA splicing, and degradation of the complex). This increases the transcription activity, which is linked to gene expression.
One example of the corepressors is histone deacetylase which catalyzes the deacetylation of acetylated lysine. This deacetylation of the lysine causes the histone complex to be positively charged at neutral pH and is able to bind to the negatively charged DNA, making these histone binding DNA region inaccessible to transcription factors, RNA polymerase and other proteins. Thus, it decreases the transcription activity. [45-48]
We will be focusing on corepressor activity as raloxifene is an estrogen antagonist.
An antagonist is a compound that fits perfectly into the binding site of the receptor. The binding of an antagonist does not bring about any biological response unlike the natural ligand and it causes the blockage of the binding site so that it is not available to the natural ligand. It has similar functional groups as the natural ligand for intermolecular interactions between amino acid residues in the binding region and most probably has extra binding regions to cause an induced fit that fails to activate the receptor. There are 4 types of antagonism: non competitive (irreversible) antagonism, competitive (reversible) antagonism, non competitive (reversible) antagonism and antagonism by umbrella effect.
e) Raloxifene as an antagonist to intracellular receptor
For Raloxifene to be an effective drug to bind to the intracellular receptor, it must be able to cross the cell membrane, meaning it must be sufficiently hydrophobic. From the structure of the drug, we could see that it resemble the rigid structure of most steroids molecules. Hence, it is able to cross the hydrophobic lipid bilayer easily and enters the cell to bind with oestrogen receptor.
Raloxifene, being a competitive antagonist , binds reversibly to the binding site. It blocks the ligand s access to binding site and causes a different induced fit that fails to activate the receptor. The level of antagonism is dependent on the antagonist binding and concentration; by increasing the concentration, the antagonism effect can be reversed.
f) Effect of raloxifene (antagonist) on estrogen receptor
The drug is known as selective estrogen-receptor modulator, which is a ligand that binds to estrogen receptor via competitive binding because it can mimic the hormone estrogen and bring about or block the estrogen action.  It can bind effectively to both ERa and ERb.
After crossing the hydrophobic lipid bilayer, the drug can bind to the ligand binding region of the estrogen receptor. The drug has similar binding interaction as estradiol to the ligand binding region of the oestrogen receptor: hydroxyl groups of benzothiophene ring of raloxifene similar to 2 hydroxyl groups on estradiol. [4, 11] However, the presence of an additional side chain (tertiary amine) can be protonated and form salt bridge interaction with Asp 351, producing antagonist activity. This causes the C-terminal a helix (H12) to change its conformation, preventing it from folding over as lid and not revealing the AF-2 (activation function-2) region of the receptor. The transcriptional coactivators cannot gain access to the binding region, thus the activation of estrogen-responsive genes is turned off. As a result, transcription is blocked. 
Figure 9: Interaction between raloxifene and estrogen receptor [11, 13]
Although this mechanism is a good hypothesis of the action of estrogen and raloxifene on estrogen receptor, the real mechanism is much more complex because not only the ligand structure has to be taken into account but also tissue and cell-specific factors. Other possible mechanisms of raloxifene action include inhibits estrogen bioavailability, decreasing the risk for breast cancer; induces apoptosis; and inhibits collagen biosynthesis, leading to decrease in collagens which interact tumour cells, thus inhibit progression of tumour. 
We will be looking into the Pharmacokinetic of the drug: absorption, distribution, metabolism, excretion.
Once administered orally, the drug is absorbed rapidly, not affected much by the food. 60% is absorbed and undergo first pass metabolism to glucuronide conjugates. Its bioavailability is quite low (2%). 
After absorption, Raloxifene and its metabolites are mostly bound to plasma proteins, albumin and a1-acid glycoprotein (95%) and is rapidly distributed around the body (2348 L/kg for single doses ranging from 30 to 150 mg and not dose dependent). 
Raloxifene is converted to its glucuronide metabolites during first-pass metabolism and does not seem to be metabolized by cytochrome P450 pathway: raloxifene-4'-glucuronide, raloxifene-6-glucuronide, and raloxifene-6, 4'-diglucuronide, see Figure 5. It has a long plasma elimination half-life of approximately 27h as raloxifene and its conjugates are interconverted by reversible systemic metabolism and enterohepatic cycling. 
Figure 5: Structure of Raloxifene and its 2 major metabolites (Raloxifene-6-B-glucuronide and Raloxifene -4 -B-glucuronide)
This glucuronidation process, which is the transfer of glucuronic acid to substrates is catalyzed by UDP-glucuronosyltransferases (UGTs) and is crucial in facilitating elimination as it results in conjugates with increased water solubility. 
It is then excreted through mostly by faeces and only a small portion is excreted by the urine (unchanged and metabolites). 
5. Effects of the drug antagonist including its side effect
We will be discussing about the clinical side effects of Raloxifene and interaction of Raloxifen with other drugs in the following part of the report. Raloxifene is initially called an antiestrogen as they antagonize oestrogen receptors  and prevent oestradiol from binding . Raloxifene is also referred to as a selective oestrogen-receptor modulator (SERM) and used in the prevention of breast cancer.  Raloxifene comes in the form of a tablet and is administered orally. It is usually taken daily with or without food.
Evista 60 mg (picture from official website of EVISTA)
Raloxifene is used to treat oesteoporosis in postmenopausal women by making bones stronger as it affects the cycle of bone breakdown and formation in the body, decreasing loss of bone tissue. It is also used to decrease the risk of invasive, estrogen receptor (ER) positive breast cancer in women who are at risk of invasive breast cancer. 
Like other drugs, Raloxifene has side effects. The side effects of Raloxifene include hot flashes (more common in the first 6 months of Raloxifen therapy), leg cramps,swelling of the hands, feet, ankles, or lower legs,flu-like syndrome, joint pain, sweating, insomnia. There are other more serious undesirable side effects of Raloxifene which may even cause death. There may be increased risk of deep vein thrombosis and pulmonary embolism with the consumption of Raloxifen. [data for side effects from official EVISTA site: http://www.rxlist.com/evista-drug-patient.htm]
Other severe side effects include sudden numbness or weakness especially on one side of the body; sudden headache, confusion, problems with vision, speech, or balance; chest pain, sudden cough, wheezing, rapid breathing, fast heart rate; unusual vaginal bleeding; breast pain, tenderness, or lump; pain or burning when you urinate; severe pain in your lower back. 
Drug-drug interactions may occur when Raloxifene is consumed. This means that if both drugs are consumed at the same time, one drug will affect the activity of the other drug. Other drugs like cholestyramine (Questran, Prevalite), blood thinners like warfarin (Coumadin) may interact with Raloxifen. The absorption and enterohepatic circulation of raloxifene will be lowered by 60% after one doze by Cholestyramine which is an anion exchange resin. Other anion exchange resins, like colestipol may have similar effects so it is unadvisable to take Raloxifene with cholestyramine at the same time. 
Raloxifene is more than 95% bound to plasma proteins.  This will decrease the bioavailability of the drug available to the target cells. It has not been determined yet whether raloxifene will affect the protein binding of highly protein-bound drugs like diazepam, diazoxide, and lidocaine.
The interaction of Raloxifene with other drugs like warfarin is not that crucial and it is not possible to have clinical impacts as the effects are slight. In vitro, Raloxifene did not interact with the binding of warfarin. To test whether there is a possibility that Raloxifene and warfarin can interact, 15 healthy postmenopausal women each were given single doses of warfarin 20mg before and during two weeks of dosage with Raloxifene 120mg/day. 
In vivo, consumption of raloxifene with warfarin decreased the clearance (CLP/F) of R- and S-warfarin by 7.1% and 14% and the oral volume of distribution (VSS/F) by 7.4 and 9.8%. 
6. Compare with Hormone Blocking Therapy, Chemotherapy, monoclonal antibodies
Treatment recommendations depend on pathological features of breast cancer. Women whose tumors are ER and/or PgR positive can have hormone therapy such as tamoxifen or raloxifene while women whose tumors are Her2 positive can use the drug Herceptin. For moderate or poor risk tumors, chemotherapy is usually recommended. Especially in younger women for ER and/or PgR negative, chemotherapy is also recommended. 
In this section of the report, we will be comparing Raloxifene with the other types of hormone therapy like aromatase inhibitiors and with other therapies like chemotherapy and monoclonal antibodies. Hormone therapy is usually used to treat hormone responsive cancers so that the cells will have insufficient oestrogen. Aromatase inhibitor differs from Raloxifene which belong to selective estrogen receptor modulators (SERMS) like tamoxifen as they cannot reduce the risk of breast cancer but they are used in the treatment of breast cancer. Aromatase inhibitors can only be used to treat breast cancer in postmenopausal women as they cannot stop the synthesis of estrogen by the ovaries of premenopausal women.
a) Aromatase inhibitors
There are two types of aromatase inhibitors: irreversible steroidal inhibitors (exemestane: Aromasin ) and non-steroidal inhibitors (anastrozole: Arimidex , letrozole : Femara ). Irreversible aromatase inhibitors inactivate aromatase by forming a covalent bond with the aromatase enzyme complex. Non-steroidal inhibitors inhibit the enzyme by reversible competition. Aromatase inhibitors work by blocking an enzyme that converts adrenal sex hormones, androgen, into estrogen in postmenopausal women.  Raloxifene can act as an antagonist to block the ER (estrogen receptor) via competitive inhibition whereas aromatase inhibitors reduce the estrogen levels in the body by directly blocking local estrogen production in the breast tumor. Aromatase inhibitors are most of the time used as second-line therapy in postmenopausal women who may have already used tamoxifen. Aromatase inhibitors may be popular as first-line therapy in the adjuvant setting(additional treatment after surgery).  This is due to early findings from an ongoing study, ATAC (Arimidex and Tamoxifen Alone or in Combination), showed that anastrozole and a combination of tamoxifen and anastrozole was found to be superior to tamoxifen alone. In ATAC, it was showed that anastrozole had a longer efficacy and higher level of safety compared to Tamoxifen initial adjuvant therapy for postmenopausal women with hormone-sensitive early breast cancer 
Letrozole (Femara ) anastrozole: Arimidex
Structure from http://www.drugbank.ca/drugs/DB01006 ;http://www.drugbank.ca/drugs/DB01217
b) Selective estrogen receptor modulators (SERMs)
There are two groups of clinically available SERMs: triphenylethylenes and benzothiophenes. Tamoxifen is a triphenylethylene and is used in treatment of breast cancer and its prevention by reducing the risk of breast cancer in women who are at high risk for breast cancer. On the other hand, Raloxifene is a benzothiophenes which was developed specifically to avoid the uterotropic effects of other SERMs like Tamoxifen as using Tamoxifen for a long period of time will cause uterine cancer seen from estrogen agonism in the uterine .
The different SERMs are either complete estrogen antagonists or partial agonists at the ER when there is a lack of estrogen in animals. As shown in the model of an estrogen-treated rat which was used to find out the uterine liability of ligands for estrogen receptor, Raloxifene blocks the uterotrophic effects of estrogen with an ED50 of 0.3mg/kg .The behavior of a partial agonist is shown by Tamoxifen. Tamoxifen significantly antagonize the effects of estrogen in the immature rat uterus but the highest level of the antagonism is only around 50% . This is due to the fact that at higher doses, Tamoxifen having a stimulatory effect on uterus restricts further suppression of the response induced by estrogen.
In clinical trials, both Tamoxifen and Raloxifene have decreased the frequency of the occurrence of breast cancer in selected populations. Raloxifene can also be used in the prevention of osteoporosis unlike Tamoxifen. A fourfold increase in endometrial carcinoma in women over age 50 is observed when Tamoxifen is used but this risk was not observed in younger women or observed with raloxifene [30, 58].
The comparison of Raloxifene with tamoxifen in terms of side effects and preventing invasive, non-invasive cancer and the risk of getting heart attack etc was carried out in a large trial involving postmenopausal women at high risk of breast cancer (the Study of Tamoxifen and Raloxifene, or STAR trial). Participants were postmenopausal women over the age of 35 with a risk of breast cancer of at least 1.66, as determined by the Gail model  , or a prior history of a precancerous breast condition, lobular carcinoma in situ (LCIS).
It is shown that Raloxifene had high degree of inhibition of estrogen receptor, Kd ~50pmol/l which is even higher than tamoxifen Kd ~2nmol/l. Raloxifene has IC50 value of 0.2nM, which is better as compared to tamoxifen (IC50= 200nM). However from the results from STAR trials, both tamoxifen and raloxifene had the same effects in preventing invasive breast cancer but raloxifene had less effect on the uterus and lower risk of blood clots than Tamoxifen as shown in the table 1 below.
As shown in table 2, participants in STAR who were assigned to take raloxifene had fewer serious side effects from that drug than participants assigned to take Tamoxifen. 
In the MORE (Multiple Outcomes of Raloxifene Evaluation) trial which included not only high-risk women with breast cancer, the incidence of breast cancer was 76% lesser with raloxifene than with Placebo, with an absolute risk reduction of 8 cases per 1000 women over 40 months.  The effect of raloxifene was only limited to ER positive receptors.
Raloxifene and Tamoxifen use different mechanism to prevent the growth of cancer cells. In the case of Raloxifene, the binding of coactivator is prevented and the transcription complex cannot be formed, therefore preventing transcription. In , the random appearance of a mutant receptor that would interpret an antiestrogen as an estrogen was investigated. In a Tamoxifen-stimulated tumor, the major mRNA species was the mutant receptor Asp351Tyr. Cells with a mutant receptor would have a growth advantage and the current results from  confirmed and it was found out that a Tamoxifen-stimulated breast tumor can improve the estrogenic properties of an antiestrogen. Tamoxifen-stimulated growth of the tumor may be due to selection of cells with high levels of coactivators. Transcription rate is high in the cells that are resistant to tamoxifen as they respond to the high levels of coactivators so and continue to grow at an uncontrollable rate instead of their growth being controlled by Tamoxifen. However, the activity of Raloxifene would not be affected by the mutation as shown in Fig 5. To solve this problem of resistance to tamoxifen, we can use raloxifene which prevents the coactivators from binding as even if high levels of coactivators is present, coactivator is prevented from binding in the presence of Raloxifene.
Fig 5 from 
Overall, Raloxifene is better than Tamoxifen with considerations in the side effects and the risk of uterine cancer of both drugs and furthermore, Raloxifene can be used on Tamoxifen failure in the presence of a mutant receptor discussed previously.
Chemotherapy refers to the use of cytotoxic drugs (e.g. CMF- cyclophosphamide, methotrexate and fluorouracil) to destroy breast cancer cells. The drugs are usually administered usually via the intravenous (IV) route whereas raloxifene is consumed orally. This differs from Raloxifene as Raloxifene can only reduce the risk of breast cancer and cannot destroy breast cancer cells. Chemotherapy can be used for adjuvant therapy, non-adjuvant therapy and to treat metastatic disease. Neo-adjuvant therapy may be given as a first line treatment before surgery or radiotherapy to reduce the size of the tumor that cannot be operated in its current state so as to allow it to be surgically removed. Adjuvant therapy is done after primary therapy like surgery to increase the chance of long-term disease-free survival as it is used to kill any cancer cells that may have spread but are not detected by imaging or laboratory tests.
Combination chemotherapy, which is the treatment of using more than one anti-cancer drug simultaneously , can be carried out and this treatment differs from hormone treatment using raloxifene as it only uses one drug at one time.
The disadvantage of undergoing chemotherapy is that the chemotherapy drugs does not specifically target on uncontrolled rapidly dividing cancer cells and will cause rapidly dividing drugs to stop dividing and self-destruct. Other cells in the body that divide fast enough can be affected by the chemotherapy drugs. This will result in undesirable side effects like hair dropping, mouth sores, nausea, diarrhea and constipation.
d) Monoclonal antibody therapy
Monoclonal antibody therapy is highly specific with low toxicities and it uses monoclonal antibodies [need to find new reference] that are made by similar immune cells that are all clones of a unique parent cell and designed to seek out as targets specific substances recognized by the immune system(antigens). Trastuzumab (Herceptin ) is a commercially available drug used to treat metastasis cancer and is used in the treatment of early stage breast cancer that is Human Epidermal growth factor Receptor 2-positive (HER2+) and has spread into the lymph nodes, or is HER2+ but has not spread into the lymph nodes. [find new reference] This differs from raloxifene as it can be used to treat early stage breast cancer and only effective in tumors which overexpress the receptor whereas raloxifene can only be used to prevent breast cancer in postmenopausal women who are at risk of breast cancer.
Trastumab targets the protein coded by HER-2/neu gene  and attaches directly to the HER2 receptor which belongs to the epidermal growth factor receptor family of tyrosine kinases. HER-2 receptors are embedded in the cell membrane and switch genes on and off when it receives molecular signals from outside the cell to inside the cell. When trastuzumab binds to defective HER2 proteins, the HER2 protein will not cause cells in the breast to reproduce in an uncontrollable rate. Another difference between raloxifene and trastumab is that by the food and drug administration, trastumab has to be administered with chemotherapy drugs like paclitaxel but can also be administered alone in metastatic breast cancer while raloxifene can be administered alone. Trastumab is an antibody which causes the immune system to destroy the affected cell  whereas raloxifene is an antagonist which binds to oestrogen receptor.
7. Propose alternatives
Another way of synthesizing the compound can be done, giving better yield because of lesser steps.
For intermediate 6, DIBALH, -78oC, THF can be used to reduce the ester group to an aldehyde, which can then undergo Grignard reaction with intermediate 8 to obtain the final product. Although it is considerably easy to carry out the reaction under the desired conditions in lab by adding acetone to dry ice, it is hard to carry out on a large scale manufacturing basis because it is hard to maintain the reaction at -78oC for reduction of the ester. An alternative method would be to use a 2 steps method to convert the ester group to an aldehyde: 1) Add lithium aluminium hydride in ether to reduce the ester group to alcohol; 2) Add pyridinium chlorochromate in dichloromethane to oxidize the alcohol group to aldehyde. By this method, the problem of carrying it out under conditions difficult to maintain at manufacturing level can be overcome.
Tumors require sufficient nutrients to grow more than 1-2nm in size and this is possible with angiogenesis which is the the process of formation of new capillary system, will connect existing blood vessels to the tumor. To prevent tumors from growing, we can use anti-angiogenesis drugs. Currently in the market, bevacizumab (avastin) is available as an anti-angiogenesis agents. Avastin blocks VEGF leading to the death of cells and making it more vulnerable to chemotherapy.However, Avastin can only binds to only one angiogenesis promoting agent . it is possible to develop another anti angiogenesis agent to bind more angiogenesis promoting agents. A natural antiangiogenic protein, endostatin is found. Endostatin (a fragment) of a protein, collagen 18 ,which is found in all blood vessels and secreted by tumors. It has been shown to inhibit the growth of blood vessels necessary for tumor development. It may be possible to use endostatin as a lead compound. By using anti-angiogenesis agents, the tumor can be below 1-2nm in size, it maintains a balance with its surrounding tissues - cell multiplication and cell death which ensures that the tumor remains at the same size.
Combination therapy can be used as a means of treatment for breast cancer patients. A specific drug treatment may not work for certain patients and perhaps, using a combination of drugs will be able to treat breast cancer. Furthermore, cancer is usually an accumulation of mutations.
Other protein target other than estrogen receptor
Studies on other proteins that lead to the cells dividing to form cancer have been undertaken. One of which is on MYB protein. MYB turns off body s self-denfense mechanisms which cause the cells to undergo apoptosis, allowing the cancer cells to keep dividing. By blocking MYB in the presence of DIA, which are molecules which usually either turn the breast cancer cells into different, non-growing cell types, or kill them, the cells will not divide uncontrollably and they can undergo cell suicide.
Targetting DNA instead of protein
Instead of having protein as the drug target, we propose that nucleic acid could be the drug target as well. This might be a better alternative because the nucleic acid is the genetic blueprint for the synthesis of the protein. An inhibition of the nucleic acid sequence would lead to an amplified inhibition of the production of many copies of the specific protein. This would appear to be a better choice for treatment of cancer as the uncontrolled gene expression can then be modulated. On top of that, nucleic acid can be used for overcoming the problem of drug resistance by regulating the expression of multidrug resistance gene and increasing the sensitivity of the resistant cells to drugs used in chemotherapy. 
As cancer is caused by genetic mutation in genes that are involved in cell growth, proliferation and repair of DNA, we explore the probability of having an inhibitor for those specific genetic mutations. As stated by Genetics Home Reference, the mutation of the BRCA1, BRCA2, CDH1, PTEN, STK11, and TP53 genes increase the risk of developing breast cancer. On top of them, AR, ATM, BARD1, BRIP1, CHEK2, DIRAS3, ERBB2, NBN, PALB2, RAD50, and RAD51 genes are also associated with breast cancer. 
We explore the probability of using antisense therapy, which is the usage of oligonucleotides to bind to and block specific sequences on mRNA, inhibiting the translation process and protein synthesis. By inhibition of the mutated gene sequences, it is possible that the synthesis of a mutated protein could be blocked and the undesired downstream effects could be blocked. 
A problem with this therapy strategy is that cancer is caused by a combination of genetic mutation so by targeting the mutation on a specific gene is not entirely effective in controlling the uncontrolled cell division. A possible way of solving this particular problem is by combining the oligonucleotides for different mutated gene sequences together so that they can inhibit the whole gene sequences. However, a longer nucleotide sequence would be poorly absorbed and susceptible to metabolism. Inhibition of gene sequences that code for other functional proteins that lie between the mutated gene sequences is also a potential problem that could have drastic effect on the proper functioning of the body system. 
A more probable way would be by using the short segments of double stranded miRNA excised by RNase III Drosha and DGCR8. They then move from nucleus to cytoplasm and are cleaved by Dicer to form miRNA. It can bind to RNA induced silencing complex which unravels the strands to produce siRNA. 1 strand is discarded and the other one which is still bounded to the protein can bind to mRNA via complementary base pairing. siRNA is involved in post-transcriptional modification because they can bind to the mRNA via 3 untranslated region. Complementary base pairing that are perfect or near perfect results in the cleaving and degradation of the mRNA, while partial base pairing results in inhibition and silencing of the gene. [50, 51]
Figure 10: miRNA biogenesis, leading to the production of siRNA (in this diagram represented as mature miRNA) which can cause degradation of mRNA or silencing of the gene 
There are 2 ways of controlling the tumour growth by siRNA method: 1) Replicate the action of endogenous microRNA to allow siRNA to decrease the uncontrolled rate of cell growth or cause cell death or inhibit endogenous microRNA that causes cancer.  2) Reintroduce underexpressed endogenous miRNA to overcome repression of miRNA, which is frequently seen in cancer development (reverse approach). 
It is a better method because it is possible to construct miRNA with a combination of binding sites that competitively inhibits the binding region of an entire endogenous miRNA family. This would help solve the most major problem which is that cancer is a collection of mutations and not just one gene but rather a few genes have to be inhibited for the cell proliferation to be controlled. On top of this, it is also more stable and less toxic in inhibiting endogenous miRNA. 
However, it is a great challenge in ensuring that the siRNA is able to arrive at and enter the target cells as well as the stability of the siRNA against metabolic activity .
It can be used together with chemotherapy or radiation to inhibit the genes involved in drug resistance, making the treatment more effective. It might be able to eliminate the cells supporting the growth, thus indirectly controlling the growth of the tumour. 
Another possible method of targeting the genes is using genetic engineering, which can knock out the mutated genes that could later cause cancer growth and the right gene sequences could be inserted in.  This would be a good method for preventing cancer for those with genetic predisposition. The position of cleaving and inserting of genes should also be precise to prevent further mutation by inserting into functional genes. However, the most major problem remains to be that cancer is a collection of mutation and not just one gene but rather a few genes might have to be knocked out for the cell proliferation to be controlled.
As research on the usage of nucleic acid as the target continues, it might be possible that the challenges related to the above therapies could be overcome one day.