*2.8. Parthenolide*

Parthenolide (**10**) (Figure 9) has the same chemical formula as inuviscolide (**6**) (C15 H20 O3) and is the best known sesquiterpene lactone in the germacranolide class. It was isolated for the first time from feverfew leaves and flowerheads (*Tanacetum parthenium* (L.) (Sch.Bip.), a plant known in traditional Chinese medicine for centuries to treat various ailments. Among them, it is used to relieve fever, pain of di fferent etiologies such as migraine and rheumatoid arthritis and even to treat insect bites [171].

**Figure 9.** Structure of parthenolide (**10**) and its derivatives DMAPT (**11**) and HMPPPT (**12**).

Compound **10** has been applied as an anti-inflammatory through specific inhibition of the signal proteins IKK2, STAT3, and MAPK, along with the activity and expression of many inflammatory mediators including COX, which is involved in the NF-κB mediated proinflammatory signal transduction pathway [172]. It also exhibits antitumoral activity by proliferation inhibition in various cancer cell types, including prostate, pancreas, cervical, breast, lung, colorectal, glioblastoma, multiple myeloma, and leukemia [8,173]. There are numerous reviews describing the outstanding anti-inflammatory and antitumor activities of parthenolide, its analogs, and derivatives upon di fferent pathways in human cancer cells [8,174–178].

Parthenolide (**10**) has a multitarget action mechanism. It triggers EGF receptor phosphorylation, interferes with AP-1 [179] and the signal transducer and activator of transcription 3 (STAT3) [180] and induces activation of c-Jun N-terminal kinase (JNK) [181]. Furthermore, the molecular mechanisms of parthenolide action are strongly associated with DNA-binding inhibition of two transcription factors

NF-κB [182,183], as well as the proapoptotic activation of p53, along with reduced glutathione (GSH) depletion [184]. Parthenolide rapidly induces ROS generation [185,186], and lowers histone deacetylase activity (HDAC) [187] and DNA methyltranspherase 1 (DNMT1) [188]. Additionally, parthenolide can interfere with microtubule function through tubulin binding [189].

Recent advances regarding in vivo therapeutic applications of parthenolide (**10**), mainly focused but not limited to anticancer and anti-inflammatory activities, are discussed below, together with the synergistic e ffects and toxicity of compound **10** and some derivatives.

One of the advantages of parthenolide (**10**) is its cancer stem-cell selectivity while remaining non-cytotoxic to non-tumor cells [190,191]. In this case, the molecular mechanism of parthenolide involves induction of apoptosis through mitochondrial and caspase signaling pathways, and also an increase in the cytosolic concentration of calcium, cell cycle arrest, and inhibition of metastasis [178,192].

Cholangiocarcinoma (CC) is an intrahepatic bile duct carcinoma with a poor prognosis due to being chemoresistant. Parthenolide induced oxidative stress-mediated apoptosis in CC cells and the Bcl-2-related family of proteins modulated that susceptibility [193]. Moreover, intraperitoneal injection of parthenolide at 4 mg/kg caused significant inhibition of tumor growth and angiogenesis in the xenograft model [194]. Recently, Yun et al. [195] demonstrated that low concentrations of parthenolide (5–10 μM) suppressed HO-1 expression, enhancing oxidative stress by the PKCα inhibitor Ro317549 (Ro) through inhibition of Nrf2 expression and its nuclear translocation. The e ffects of parthenolide (**10**) and Ro at 2.5 mg/kg on tumor growth were tested using a xenograft nude mouse tumor model with subcutaneously implanted ChoiCK and SCK cells. This assay indicated that their combined application more e ffectively inhibited cancer cell growth inhibition as compared to treatment with either compound by itself. Furthermore, the e ffect of parthenolide on the development of colitis-associated colon cancer (CAC) was investigated using a murine model of azoxymethane (AOM)/dextran sulfate sodium (DSS) induced CAC. This study showed that parthenolide administration (**10**) at 2 and 4 mg/kg can significantly inhibit the inflammation-carcinoma sequence and be crucial in experimental CAC regulation. The mechanism of action involves decreased NF-κB p65 expression levels blocking phosphorylation and subsequent degradation of κBα inhibitor (IκB α) [196]. The authors conclude that parthenolide (**10**) could be a novel chemopreventive agen<sup>t</sup> for CAC treatment [196].

Oral cancer is one of the five most common cancers worldwide. Chemoprevention is a new approach to cancer research, focusing more on the prevention, suppression, and reversal of the carcinogenic process by the use of natural plant products and/or synthetic chemical compounds. Thus, Baskaran et al. [197] tested the chemopreventive potential of parthenolide in DMBA-induced hamster buccal pouch carcinogenesis (DMBA, 7,12-dimethylbenz[a]anthracene). Oral administration of parthenolide (**10**) at 2 mg/kg b.w. completely prevented tumor formation and significantly reduced the nefarious histopathological changes. In addition, the parthenolide treated group showed significant improvement regarding antioxidants, detoxification enzymes and lipid peroxidation.

Glioblastoma, or glioblastoma multiforme (GBM), is the most aggressive type of brain cancer and is very di fficult to treat. Nakabayashi and Shimizu [198] examined the e ffect of compound **10** on tumor growth using a xenograft mouse model of glioblastoma, administering it intraperitoneally (10 mg/kg/day) for 22 days. It significantly inhibited the growth of transplanted glioblastoma cells with respect to the control group.

Zhang et al. [199] demonstrated that a high parthenolide dose (8 mg/kg/day) impedes initiation of experimental autoimmune neuritis (EAN), an animal model for peripheral nervous system acute inflammatory disease. This is achieved by parthenolide suppressing TNFα, IFN-γ, IL-1β and IL-17 pathways and quickly decreasing Th1 and Th17 cells in the early stages. Although this anti-inflammatory effect is short-lived, compound **10** also suppresses late-stage recovery of EAN models, along with inhibiting the apoptosis of inflammatory cells. Such results sugges<sup>t</sup> that parthenolide is not suitable for nervous system autoimmune disease treatment.

Nitric oxide (NO) plays a key role in the etiopathology of central nervous system (CNS) diseases like multiple sclerosis (MS). It has been proposed that inhibition of NO synthesis could prove a relevant mechanism of action in treating multiple sclerosis and migraine. Accordingly, Fiebich et al. [200] investigated the effect of parthenolide (**10**) on iNOS synthesis and NO release using primary rat microglia. The results indicated that compound **10** prevents iNOS/NO synthesis and inhibits the activation of p42/44 mitogen-activated protein kinase (MAPK), but not IkBα degradation or NF-kB p65 activation. These results show parthenolide may be a potential therapeutic agen<sup>t</sup> in the treatment of CNS diseases.

Mechanisms of axon regeneration and optimal functional recovery after nerve injury are key to in higher animals. However, insufficient growth rates of injured axons often lead to incomplete peripheral nerve regeneration. Gobrecht et al. [201], demonstrated that a single parthenolide injection at 5 nM into the injured sciatic nerve or its systemic intraperitoneal application was already enough to significantly increase the number and length of regenerating axons in the distal nerve at three days post-lesion. This application of parthenolide (**10**) appears to act on the grea<sup>t</sup> instability of microtubules in promoting axonal growth, at least in the CNS [174]. For this reason, the efficacy of parthenolide is very promising for a therapeutic promotion of nerve regeneration, since compound application and recurrent treatments are facilitated, compared to invasive local nerve injections [202].

Pulmonary fibrosis (PF) in general and idiopathic pulmonary fibrosis (IPF) in particular, is a disease for which there is no effective therapy. In vivo studies have shown that parthenolide (**10**), via intragastric administration, inhibited the NF-κB/Snail pathway, attenuating bleomycin-induced pulmonary fibrosis. Moreover, there were significant improvements in body weight and other pathological changes associated with this disease [203].

NF-κB has been associated with the cardiovascular system; in fact, its function is related to the protection of cardiovascular tissues against injuries. However, its activation can also contribute to tissue pathogenesis, depending on the type of cells in which it is activated [204]. It is known that myocardial infarct size could be reduced up to 60% by antagonizing NF-κB activity [205]. To achieve this, parthenolide (**10**) at 250 or 500 mg/kg (b.w.) was administered intraperitoneally before reperfusion in rats, and caused a significant improvement in myocardial injury, with a reduction in the oxidative and inflammation state, consequently reducing infarct size [206]. However, Tsai et al. [207] reported that a prolonged treatment in bEND.3 cells affected Ca2þ signaling in the endothelial cells that regulate vascular tone; therefore, care should be taken on using this compound in experimental designs and clinically.

Parthenolide (**10**) has relatively poor pharmacological properties, derived from its low solubility in water and consequently reduced bioavailability, which limit its potential clinical use as anticancer drug. To increase its solubility, a series of parthenolide derivatives were obtained by diastereoselective addition of several primary and secondary amines to the exocyclic double bond [208,209]. *N*,*N*-Dimethylaminoparthenolide (DMAPT) (**11**) (Figure 9) was selected as a leader compound according to its pharmacokinetic, pharmacodynamic and bioavailability properties [209,210]. When formulated as a fumarate salt, DMPAPT is 1000-fold more soluble in water than parthenolide and maintained the anticancer activity because, DMAPT (**11**) is rapidly converted back to parthenolide in body fluids (**10**). Recently, molecular studies indicate that DMAPT has a similar action to parthenolide [192,211–213].

DMAPT has approximately 70% oral bioavailability and induces rapid death of primary human leukemia stem cells (LSCs) from both myeloid and lymphoid leukemias and is highly cytotoxic to bulk leukemic cell populations. Pharmacological studies carried out by Guzman et al. [210] using both mouse xenograft models and spontaneous acute canine leukemias demonstrate in vivo bioactivity. Indeed, DMAPT eliminates human AML stem and progenitor cells without harm to normal hematopoietic stem and progenitor cells, and eradicates phenotypically primitive blast-crisis chronic myeloid leukemia (bcCML) and acute lymphoblastic leukemia (ALL) cells. Moreover, it inhibited metastasis in a mouse xenograft model of breast cancer and enhanced the survival of treated mice [210].

DMAPT was assayed in a phase I trial against acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and other blood and lymph node cancers [12,176]. However, a year later the clinical trials were suspended [214].

Radiotherapy is widely used in cancer treatment; however, the benefits can be reduced by radiation-induced damage to neighboring healthy tissues. Morel et al. [215] demonstrated in mice that DMAPT (**11**) selectively induces radio-sensitivity in prostate cancer cell-lines, while protecting primary prostate epithelial cell lines from radiation-induced damage. Compound 11 has the advantage of being well-tolerated orally without the need to adjust the administration time to radiation exposure. Radiation-induced lung fibrosis is considered a critical determinant for late normal tissue complications. Therefore, the same group [216] examined the radioprotective e ffect of DMAPT (**11**) on fibrosis in normal tissues, according to the image-guided fractionated radiotherapy protocols used clinically. The results obtained show that DMAPT reduced radiation-induced fibrosis in the corpus cavernosum of the rat penis (98.1%) and in the muscle layer around the bladder (80.1%), and also the tendency towards reduced collagen infiltration into the submucosal and muscle layers in the rectum. They concluded that DMAPT could be useful in providing protection from the radiation-induced side e ffects such as impotence and infertility, urinary incontinence and fecal urgency resulting from prostate cancer radiotherapy [216].

On the other hand, radiation-resistant prostate cancer cells often overexpress the transcription factor NFκB. Mendonca et al. [217] sugges<sup>t</sup> that DMAPT might have a potential clinical role as radio-sensitizing agen<sup>t</sup> in prostate cancer treatment. This conclusion is based on the finding that combined treatment of PC-3 prostate tumor xenografts with oral DMAPT and radiation therapy significantly reduced tumor growth, when compared to those treated with either DMAPT or radiation therapy alone.

Li et al. [218], obtained a novel parthenolide derivative, HMPPPT, a 13-substituted derivative ((3 *R*,3a*S*,9a *R*,10a *<sup>R</sup>*,10b*S*,*<sup>E</sup>*)-3-((4-(6-hydroxy-2-methylpyrimidin-4-yl)piperidin-1-yl)methyl)-6,9a-dimethyl-3a,4,5,8,9,9a,10a,10b-octahydrooxireno [2,3:9,10]cyclodeca[1,2-b]furan-2(3 *H*)-one) (**12**) (Figure 9), with better bioavailability and pharmacological properties than DMAPT. In vitro studies pointed to compound **12** as the most promising derivative, from safety profile and ADME property standpoints. The newly identified compound was shown to have pro-oxidant activity and in silico molecular docking studies with components of the NF-κB pathway also supporting a pro-drug mode of action. This mechanism included release of parthenolide and covalent interaction with one or more proteins involved in that pathway [218]. The in vivo oral bioavailability study of compound 12 in murine PK at 10 mg/kg indicated that it has advantageous pharmacological properties and therefore can be considered an agen<sup>t</sup> to be considered in therapy against drug resistant chronic lymphocytic leukemia [218].

In the last few years, nanotechnology has provided many selective strategies for the detection and treatment of cancer, overcoming the problems associated with conservative cancer diagnosis and therapy. In this context, the development and testing of parthenolide (**10**) nanoencapsulation and derivatives is a way to enhance its potential to provide e ffective pharmaceutical products for clinical use and resolve drawbacks such as low bioavailability [219–222]. Accordingly, several patents for elaboration of parthenolide and its derivatives in nanocarriers, and various pharmaceutical preparations combined with other products, have been recently registered in China: patents N◦ CN 110292640, 2019; N◦ CN108721276, 2018; N◦ CN1087211330, 2018; N◦ CN106366068, 2017; N◦ CN 109276553, 2017.

In recent years, parthenolide has been suggested for use in combination therapy with other anticancer agents, to overcome obstacles in the treatment of cancer, such as a) di fferent types of cancer cells, b) resistance to chemotherapy, and c) drug toxicity to normal cells.

TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand) is now being developed as a promising natural immunity-stimulating molecule for clinical trials in cancer patients. However, various malignant tumors are currently resistant. Kim et al. [223] investigated how parthenolide (**10**) sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis. For this, HT-29 (TRAIL-resistant) and HCT116 (TRAIL-sensitive) cells were treated with compound **10** and/or TRAIL. The results revealed that parthenolide (**10**) increases induced apoptosis and upregulates DR5 protein level and surface

expression in both cell lines, suggesting that combined therapy with TRAIL is a good strategy to overcome the resistance of certain CRC cells.

Pancreatic cancer is a common malignancy with high occurrence worldwide, and a poor survival rate. Recent research indicates that combination therapy with DMAPT (**11**) can enhance the antiproliferative effects of gemcitabine in pancreatic cancer cells in vitro and in vivo [224]. Yip-Schneider et al. [225] showed that celecoxib (a cyclooxygenase 2 (COX-2) inhibitor) at 50 mg/kg/day combined with DMPTA at 40 mg/kg/da has a significant inhibitory effect on tumor invasion of adjacent organs and metastasis in pancreatic cancer induced in Syrian golden hamsters. It reduced NFκB activity, and expression of prostaglandin E2 and its metabolite. They also demonstrated that compound **11** (40 mg/kg/day) in combination with sulindac (20 mg/kg/day) and gemcitabine (50 mg/kg twice weekly) can delay or prevent progression of premalignant pancreatic lesions) in a genetically engineered mouse model of pancreatic cancer [226]. Likewise, they demonstrated that DMAPT (**11**) (40 mg/kg/day) with gemcitabine (50 mg/kg/day) considerably improved average survival, lowering the frequency and multiplicity of pancreatic adenocarcinomas. This combination also significantly reduced tumor size and the incidence of metastasis into the liver [227].

Parthenolide exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell-line, however its success is scarce at low doses. In order to overcome this difficulty, Carlisi et al. [228] tested a histone deacetylase inhibitor, SAHA (suberoylanilide hydroxamic acid), which synergistically sensitized MDA-MB231 cells to the cytotoxic effect of parthenolide (**10**). It is noteworthy that treatment with parthenolide alone increased the survival of cell pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, a protein regulating the expression of antioxidant proteins that protect against the oxidative damage triggered by injury and inflammation, while treatment with SAHA alone activated autophagy.

A phase 2 clinical trial indicated that actinomycin-D (ActD), a polypeptide antibiotic that intercalates to DNA and inhibits mRNA transcription in mammalian cells, could be a potent drug against pancreatic cancer. However, it is not a good candidate due to toxicity issues. Thus, given the modes of action of DMPTA and Actinomycin-D (ActD), Lamture et al. [229] postulated that combining both drugs would result in synergistic inhibition of Panc-1 pancreatic cancer cell growth, since the inhibitory activity of DMAPT on FkB would enhance apoptosis induction by ActD, through phosphorylation of c-Jun. Indeed, the combination of these two drugs produced a higher cell-death percentage than each drug alone.
