**2. Non-oncology drugs repurposed for breast cancer (preclinical data)**

## **2.1 Aspirin**

Aspirin was originally discovered in 1897 and was first commercialized as an analgesic. It has been utilized as an anti-inflammatory medication and for managing arterial and venous thrombosis [12]. Recent research has sparked interest in the usage of Aspirin for the prevention of various cancers. There are compelling evidences authenticating that regular use of low doses of aspirin results in a significant reduction in the occurrences and mortality of various cancers [13–17]. The possibility that Aspirin has an anticancer benefit has received considerable interest nowadays, with a lot of research being done to figure out how successful it is in the prevention of colorectal cancer [18], lung cancer [19], gastric cancer [20], prostate cancer [21], and many other cancers including breast cancer. Because of the effect of Aspirin in several biological processes such as inhibitory effect on angiogenesis [22], cancer cell metastasis [23], causing cell apoptosis [24], etc., it is reasonable to predict that Aspirin will be beneficial when employed as an additional alternative treatment option for cancer patients. Aspirin directly inhibits the activity of the enzyme cyclooxygenase (COX-2) and thereby impedes the synthesis of prostaglandin E2 (PGE-2), which leads to cancer cell death [25]. Recent research also suggests that Aspirin may mediate anticancer potential through COX-independent pathways such as inhibition of NFκB [26], downregulation of survivin [20], targeting AMPKmTOR signaling [27], Wnt signaling cascade [28], etc.

A study was conducted by Dai et al. reported that Aspirin possesses antiangiogenic and anti-metastatic potential in MDA MB 23 cell line by directly binding to the enzyme heparinase. The results were further confirmed *in vivo* experimentation [23]. Heparinase is an endo-β-D glucuronidase that is specific to heparin sulfate. It dissolves heparin sulfate chains of proteoglycans on the cell surface and extracellular matrix (ECM) that consequently contributes to the degradation of the extracellular matrix that further assists tumor invasion and metastasis [29]. Further, heparin also facilitated the release of angiogenic factor, vascular endothelial growth factor (VEGF) blocked by aspirin-mediated heparin inhibition [23]. Breast cancer cell lines (MDA MB 231 and MCF-7) showed a dose-dependent inhibitory effect on growth after treatment with Aspirin. The Aspirin further restricts the migration of these cells by preventing epithelial to mesenchymal transition through suppression of various mesenchymal markers such as vimentin and increasing expression of various epithelial markers such as Keratin-19 and E-cadherin.

Further inhibitory effect of TGF-β/SMAD4 signaling, as evident from decreasing the production of SMAD proteins, also contributes to the anti-metastatic potential of Aspirin [30]. In another study, Choi et al. demonstrated the effect of Aspirin in the MCF-7 cell line. It was observed that Aspirin alters the complex formation between Bcl-2 and FKBP38 and leads to the nuclear translocation of Bcl-2 and phosphorylation that causes its activation, contributing to its inhibitory effect on MCF-7 cell proliferation and also triggers apoptosis in cell lines [31]. In combination with exemestane, Aspirin showed synergy in inhibition of cell proliferation. Significant arrest in the G0/G1 phase was observed along with a more detrimental effect on COX-1 and Bcl-2 expression than individual therapy [32]. In addition, when combined with tamoxifen (which is used as a drug of choice for the estrogen receptor positive BC), it downregulates the level of cyclinD1. Subsequently, it arrests the cell cycle in phase G0/G1. In the same study, authors also reported that Aspirin inhibits the ER + ve BC cells growth and overcomes the resistance to tamoxifen in MCF-7/ TAM cell line. Study demonstrated a new way to treat ER + ve BC in combination therapy of Aspirin and tamoxifen [33].

#### **2.2 Metformin**

Metformin (1,1-dimethyl biguanide hydrochloride) is a well-recognized biguanide derivative and has a long history of usage in managing type 2 diabetes (T2D). Because of the outstanding ability to lower plasma glucose levels, metformin has become the primary drug for managing T2D [34]. The drug was firstly approved in 1958 in the United Kingdom, and this decade-old drug is in the WHO's list of essential medicines [35]. Metformin belongs to the category of successful repurposed drugs and advanced into the clinical trials Phase 3/4 for its use in the

*Breast Cancer Drug Repurposing a Tool for a Challenging Disease DOI: http://dx.doi.org/10.5772/intechopen.101378*

prostate, oral, breast, pancreatic, and endometrial cancers [6]. Various preclinical and clinical examinations have demonstrated the effectiveness of metformin in the treatment of various malignancies such as pancreatic cancer [36], gastric cancer [37], blood cancer [38], etc. A meta-analysis study on diabetic patients with breast cancer concluded that patients who were treated with metformin and neoadjuvant therapy had a higher pathological complete response rate (24%) compared with patients not undergoing metformin treatment (8%) [39]. Another meta-analysis study demonstrated 65% survival improvement when compared with control [40]. Metformin has increased the survival opportunity in type 2 diabetic patients suffering from invasive breast cancer [41]. Study also suggested that patients on metformin demonstrate improved in the survival and response to treatment [40]. The metformin uptake is mediated by the OCT1 in BC cells [42], which is reported to play important role in the BC cells as an anticancer activity [43]. Upon entry into the cells, it leads to increase apoptosis, anti-proliferative, anti-angiogenic, which seems to be mediated by the mTOR, Akt/MAPK pathway [44]. Study conducted by Shi et al., established that metformin can also inhibit the expression of the COX-2, suggested the potential of metformin in combination with others COX-2 inhibitor [45]. Low cost and stability of metformin make it a good candidate for the treatment of cancers when compared with available treatment options [46].

## **2.3 Itraconazole (ITC)**

Itraconazole, a triazole antifungal drug, is a well-tolerable agent that is extremely effective against a wide range of fungal infections. Itraconazole is a highly potent and effective antifungal agent due to its active metabolite, hydroxyitraconazole, which also has significant antifungal action [47]. Itraconazole blocks ergosterol synthesis in the fungal cell membrane by inhibiting the enzyme 14α-demethylase and suppressing their growth [48]. It has emerged as a potent anticancer agent because of its ability to overcome chemoresistance prompted by P glycoproteins, altering various signaling pathways such as hedgehog (Hh) signaling cascade, Wnt/β-catenin pathway in cancer cells, and also preventing angiogenesis and lymphangiogenesis [49]. Itraconazole has been shown to have the ability to eliminate cancer cells by disrupting Hh signaling [50]. In invertebrates, the Hh signaling cascade is responsible for the regulation of complicated developmental processes. However, aberrant activation of this pathway plays a crucial role in carcinogenesis and cancer maintenance and contributes to chemoresistance, thus, targeting this pathway offers the potential therapeutic possibility [51]. Itraconazole was able to exhibit cytotoxicity in breast cancer cell lines by influencing mitochondrial membrane potential through induction of apoptosis, decreasing expression of Bcl-2, and enhancing the caspase activity. Itraconazole also promoted autophagic cell death via elevation of LC3-II expression, degradation of P62/SQSTM1, formation of autophagosomes. Hedgehog signaling is an important regulator of apoptosis and autophagy. Hence, inhibition of this signaling by Itraconazole results in cytotoxicity, tumor shrinkage, apoptosis, and autophagy in breast cancer both in *in vitro* and *in vivo* investigations [50, 52]. Anticancer activity is also reported in esophageal cancer, mediated by downregulating the HERK/AKT pathway [53]. A pilot study with 13 participants demonstrated that increased levels of Itraconazole in plasma were associated with the increased level of thrombospondin-1, angiogenesis inhibitor.

Additionally, the level of other growth factors such as fibroblast growth factor (FGF) and placenta-derived growth factor also decreased without any direct association with the Itraconazole [54]. When administered in combination with other cytotoxic agents, Itraconazole increased the response rate [55]. Researchers are trying various ways to enhance the anti-neoplastic activity of itraconazole. One such example is the development of the modified lipid nanoparticles having Miltefosine (subtherapeutic dose), called M-ITC-LNC (Membrane additive itraconazole with lipid nanoparticles (Miltefosine). The results from the cytotoxicity studies demonstrated that the anticancer activity and selectivity significantly increased in MCF-7 BC cells compared with the ITC-solution and ITC-LNC without modification [56]. In another study, itraconazole was co-delivered with the doxorubicin by liposome (coated with the Pluronic P123), resulting in the increased anti-neoplastic activity in BC [57]. The combination of the verapamil and ITC with 5-FU decreased cell survival and proliferation.

Moreover, ITC and 5-FU are more effective in the treatment of BC [58]. Administration of the Itraconazole with erlotinib (tyrosine kinase inhibitor) increased the AUC and Cmax by 10.8 and 2.78-fold, respectively, without any SAE [59]. Abovementioned all the studies reveal the potential of Itraconazole alone or in combination with other anticancer agents to treat BC.

## **2.4 Simvastatin**

Simvastatin belongs to the class of statins and is a well-explored hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that reduces cholesterol biosynthesis initially used to reduce cholesterol biosynthesis marketed in 1988 [60]. Clinical data suggest that statins are effective in BC management. Statins amplify tumor cell death and radiosensitivity in various cell lines, inhibit invasion and proliferation, and show anti-metastatic activity. Clinical trials conducted on breast cancer (inflammatory and TNBC) patients also favored these observations by representing improved mortality benefits for patients on statins [61, 62]. In the same context, Simvastatin is the most explored statin to explore the role of statins in cancer. Simvastatin targets the transcription factor NFκB that reduces the expression level of anti-apoptotic protein Bcl-xL, concomitantly inhibits the expression of anti-proliferative and proapoptotic tumor suppressor PTEN and hence inhibiting the growth of breast cancer cells. The elevation of PTEN expression results in the suppression of Akt phosphorylation. Akt activity is upregulated in many cancers by increasing cancer cell survival, inhibiting apoptosis, and increasing proliferation. Therefore, Simvastatin substantially decreased Akt phosphorylation concurrently with the reduction in expression of anti-apoptotic protein by dysregulation of NFκB, thus showing the anticancer activity against BC [63]. On administration of Simvastatin, the expression of PTTG1 (pituitary tumor-transforming gene 1) was also reduced in a dose-dependent manner in the MDA-MB-231 cell line. PTTG1 is the important gene involved in the invasion and metastasis of BC [64]. In the same cell line (MDA-MB-231), Simvastatin leads to fragmentation of the cell's nuclei, subsequently inducing apoptosis. It also enhanced the level of ROS in a dose-dependent manner, which causes oxidative stress and further DNA damage [65]. Apoptotic effects were due to the increased expression of miR-140-5p in a dose-dependent manner mediated by the activating transcription factor NRF1 [65]. Apart from the MDA-MB-231 cell line, Simvastatin effects were also explored in other breast cancer cell lines such as T47D, BT-549, and MCF-7, showing apoptotic inducer anti-proliferative activity [66, 67]. In *in vivo* studies with DMBA (dimethyl-Benz(a)anthracene) induced breast cancer rat model, Simvastatin reduced the tumor volume by around 80% [68]. Karimi et al. also explored its activity in breast cancer mice model and reported improved mortality and tumor volume compared with control [69]. Although Simvastatin's lipophilic nature makes it a good candidate for the BC treatment, the researcher tried to develop nano formulations to improve the delivery in a targeted specific manner and reduce the non-target side

*Breast Cancer Drug Repurposing a Tool for a Challenging Disease DOI: http://dx.doi.org/10.5772/intechopen.101378*

**Figure 3.**

*Simvastatin acts via blocking p50–65 leading to activation of PTEN, which inhibits PI3K-Akt axis leading to inhibition of cell growth, division, survival, migration, and proliferation.*

effects. Detailed mechanism of cell growth inhibition, division, survival, migration, and proliferation by Simvastatin is presented in **Figure 3**.

In the same series, Sed et al. used nanoparticles made of superparamagnetic iron oxide to Simvastatin delivery with enhanced anticancer activity in the PC-3 cell line. This action is mediated by inducing apoptosis and cell cycle arrest in the G2 phase [70]. Researchers from another lab developed poly D, L-lactide-co-glycolide (PLGA) with cholic-acid-based nanoparticles for Simvastatin release in a sustained and controlled manner for breast adenocarcinoma treatment. These nanoparticles showed maintainable and more efficiently inhibit tumor growth than normal Simvastatin [71]. Other formulations such as nanocapsule [72], nanoemulsions [73], liposomes [74], and immunoliposome [75] for Simvastatin were developed with increased anticancer activity in breast cancer cells. In a randomized placebocontrolled study, Simvastatin shows a better anticancer profile with the carboplatin and vinorelbine in metastatic breast cancer [76]. Consistency in the results from both clinical and preclinical studies suggests the vast potential of Simvastatin in treating breast cancer either alone or in combination. Moreover, the development of nanoformulations also provided advantages such as enhanced cytotoxicity, lower side effects, targeted delivery over the conventional available treatment options for BC.

#### **2.5 Niclosamide**

Niclosamide, an FDA-approved anthelminthic drug used to manage tapeworm infection, has been used almost from the last half of the century and included in the WHO's list of essential medicines. Recent research suggests that niclosamide has a wide range of therapeutic uses other than treating parasitic infection. Niclosamide's


*Drug Repurposing - Molecular Aspects and Therapeutic Applications*

**Table 1.**

*Summary of the repurposed drugs for BC discussed in the chapter.*

#### *Breast Cancer Drug Repurposing a Tool for a Challenging Disease DOI: http://dx.doi.org/10.5772/intechopen.101378*

clinical application diseases include type 2 diabetes, endometriosis, neuropathic pain, bacterial and viral infections, including cancer [77]. The anticancer benefits of niclosamide have been shown in many malignancies such as colon cancer, lung cancer, prostate cancer in humans, as well as breast cancer by suppressing various cancer related pathways such as Wnt Notch, mTOR, STAT, and NFκB [78, 79]. The combinational treatment of niclosamide with cisplatin overcomes the resistance to cisplatin and induces an inhibitory effect on proliferation *in vitro* and reduced tumor size *in vivo*.

Further, niclosamide prevented the epithelial-mesenchymal transition (EMT) by suppressing mesenchymal markers such as snail and vimentin. The inhibitory effect on EMT and prevention of stem-like phenotype of TNBC by Niclosamide operate by disabling various abnormal signaling pathways such as Akt, ERK, and Src [80]. The niclosamide acts as a potent inhibitor of STAT signaling by preventing cancer cell proliferation, invasion, and metastasis by decreasing the phosphorylation of STAT3 that otherwise was found in 35% of breast cancer tissues. Furthermore, STAT3 promotes the expression of several key downstream genes involved in proliferation, cell survival, and angiogenesis in breast cancer [81]. Human monocyte cells were reduced to HUVECs in the presence of niclosamide. Niclosamide also inhibited VCAM-1 and ICAM1 protein expression in HUVECs. Niclosamide decreased HUVEC proliferation, migration, and development of cord-like structures. *In vivo*, niclosamide inhibits VEGF-mediated angiogenesis [77]. Niclosamide inhibited Wnt/Frizzled 1 signaling, mediated by the increased degradation of the Wnt co-receptor LRP-6 (low-density lipoprotein receptor-related protein 6) [82–84]. Osada et al. determined that on the administration of niclosamide, there was a decrease in Dvl2 expression, which further impeded the downstream signaling (β-catenin) [85]. Londoño-Joshi et al. reported that niclosamide administration also reduced levels of LRP6 and β-catenin in breast cancers [86]. In combination with doxorubicin, niclosamide induces apoptosis and synergistically increases breast cancer cell death. This action is mediated by Wnt/βcatenin pathway downregulation and arrest of the cell cycle by Niclosamide in G0/ G1 while both doxorubicin and niclosamide increased ROS production, thus showing cytotoxicity [87, 88]. Niclosamide also showed synergistic anticancer activity with 8-quinolinol [89]. When niclosamide is administered with cisplatin, it could inhibit the invasion and cell stemness of breast cancer cells, mediated by downregulation of anti-apoptotic protein Bcl2 [90]. In a recently published study, albuminbound niclosamide (nab-Niclo) (Albumin-based nanoparticle transport systems) was found to inhibit cell growth, induce cell death, mitochondrial dysfunction, and increase oxidative stress with DNA damage. This nab-Nicolo was appeared more effective than normal Niclosamide for BC treatment [91]. Taken together, all the data suggest that niclosamide alone and in combination with other drugs could be used for the normal BC and resistance BC all repurposed drugs for BC discussed in this chapter summarized in **Table 1**.
