Reposition of the fungicide ciclopirox for cancer treatment

Abstract

BACKGROUND:

Ciclopirox (CPX), a broad-spectrum fungicide, has been widely used to treat fungal infection on the skin and nails for decades. Recent preclinical and clinical studies have shown that CPX also possesses promising anticancer activity.

OBJECTIVE:

To summarize the patents, the pharmacological and toxicological properties, the anticancer activity, and the mechanisms of action of CPX and its derivatives as anticancer agents.

METHODS:

We searched PubMed and Google using the keywords “ciclopirox”, “cancer or tumor” and “patent”, and reviewed the literature identified.

RESULTS:

Pharmacological and toxicological profiles from preclinical and clinical studies support that systemic administration of CPX and its derivatives is feasible and safe for cancer treatment. CPX exerts its anticancer activity by inhibiting cell proliferation, inducing apoptosis, suppressing cell migration and invasion, and inhibiting angiogenesis and lymphangiogenesis. Mechanistically, CPX impacts the expression or activities of multiple signaling molecules or pathways, such as ribonucleotide reductase, Myc, DJ-1, Wnt/β-catenin, DOHH/eIF5A/PEAK1, VEGFR-3/ERK1/2, ATR/Chk1/Cdc25A, and AMPK/TSC/mTORC1. Most of these effects are attributed to iron chelation by CPX. Five patents have been retrieved: four patents on the development of CPX prodrugs to improve the water solubility and bioavailability of CPX, and one patent on the methods of bladder cancer treatment with CPX, CPX-O, or a CPX prodrug.

CONCLUSION:

CPX has a great potential to be repositioned for cancer therapy.

1. INTRODUCTION

Discovery and development of a new drug is time-consuming, laborious, and expensive (1). It takes over 10 years and costs more than US$1.3 billion to bring a new drug to market (2, 3). An alternative strategy is to “repurpose” existing drugs for new uses (4). Because of the known bioavailability, safety and toxicity in humans and animals, an off-patent drug with previously unrecognized anticancer activity could be rapidly repurposed for cancer therapy. A good example is thalidomide, which was a sedative drug and has been repurposed to treat multiple myeloma (5).

Ciclopirox (CPX) is a synthetic antifungal agent, having a broad spectrum of action against dermatophytes, yeast, filamentous fungi and bacteria (6-8). CPX has been widely used for the treatment of superficial fungal infection for over 30 years. The mechanism of antifungal action of CPX is not well understood. However, it has been proposed that the antifungal activity of CPX is primarily attributed to its chelation of trivalent metal cations such as Fe³⁺, inhibiting the metal-dependent enzymes responsible for degradation of toxic metabolites and targeting diverse metabolic and energy producing processes in the cells (6-8). Recently, CPX has been found to have considerable potential to act against many other human diseases including cancer (8), diabetes (9-11), cardiovascular disorders (12-14), and acquired immune deficiency syndrome (AIDS) (15-17). In addition, CPX exhibits protective effects on neurons as well (18-20). These findings implicate that CPX is a very promising agent for treatment and/or prevention of multiple human diseases. In this review, we focus on discussing the anticancer effect of CPX. Specifically, we will briefly review the history and literature of CPX as a fungicide, and then discuss the patents, the mechanisms of action, as well as preclinical and clinical studies of CPX and its derivatives as anticancer agents.

2. BRIEF HISTORY OF CPX AS A FUNGICIDE

CPX is a synthetic antifungal agent, which belongs to the hydroxypyridone family. Clinically, CPX is commonly used as an olamine salt, ciclopirox olamine (CPX-O) (Fig. 1), and sold worldwide under many brand names such as Batrafen, Loprox, Mycoster, Penlac, and Stieprox (5). Currently CPX is often used to treat superficial fungal infection of the skin and nails (5-7). Various topical formulations of CPX and CPX-O are available for application onto the skin and nails and into the vagina. Lotion, spray, shampoo, pessary, solution, gel and douche forms have been developed, but 1% CPX-O cream and 8% CPX acid nail lacquer are the most widely used formulations (7). The chemical name of CPX is 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridinone (CAS Number: 29342-05-0), with a molecular formula of C₁₂H₁₇NO₂, and a molecular weight of 207.27. CPX is a white or light yellow powder, with a melting point of 128–130°C (8). CPX-O (CAS Number: 41621-49-2) has a molecular formula of C₁₂H₁₇NO₂·C₂H₇NO, with a molecular weight of 268.35. Both CPX and CPX-O are hardly soluble in water, but very soluble in methanol, ethanol and dimethyl sulfoxide. CPX, as a fungicide, was first marketed in 1982, so it is an off-patent antifungal drug.

Fig 1.

Chemical structures of ciclopirox and its derivatives.

CPX possesses broad-spectrum antifungal and antibacterial activity. It can be both fungistatic and fungicidal in a broad spectrum of fungal organisms, such as dermatophytes, yeasts, dimorphic fungi, eumycetes, and actinomycetes (6-8). Its minimum inhibitory concentration range against these pathogens is commonly from 0.9 to 3.9 μg/mL, except from 1.9 to 15.6 μg/mL against molds (6-8). Also, it exerts antibacterial activity against many Gram-positive and Gram-negative bacteria (6-8). There is no difference in pharmacological aspects between CPX and its olamine salt (6-8). The antifungal activity of 0.77% CPX is roughly equal to that of 1% CPX-O, because the olamine entity has no antifungal activity (6-8).

Though CPX has been clinically used as a fungicide for decades, its antifungal mechanism is still poorly understood. It has been described that CPX inhibits the growth of fungi by disrupting DNA repair, cell division signals and structures (mitotic spindles) (21) and by interrupting the membrane transfer system (Na⁺/K⁺-ATPase) (22). It has also been proposed that CPX executes its antifungal activity by chelation of iron, as addition of iron ions (e.g. Fe²⁺ and Fe³⁺), but not other metal ions (e.g. Ca²⁺, Mg²⁺ and Mn²⁺), attenuates the antifungal activity of CPX (23). Iron is essential for the activity of many enzymes, such as glucose-6-phosphate-dehydrogenase, cytochrome c peroxidase, catalase, peroxidase, superoxide dismutase, and ribonucleotide reductase (RR) (24, 25). Hence, it is not surprising that CPX treatment interferes with mitochondrial electron transport, energy production, and reactive oxygen species (ROS) clearance, and results in DNA damage (8). The unique and multilevel mechanism of action may contribute to not only its broad-spectrum antifungal activity but also its rare cases of resistance clinically.

3. PHARMACOLOGICAL AND TOXICOLOGICAL PROPERTIES OF CPX

Early pharmacokinetics and biotransformation studies have shown that topical application of 1% CPX-O cream to human skin results in a percutaneous absorption of about 1.3% of the dose (26). Above the minimum inhibitory concentrations of CPX can be detected in the dermis in 1.5 hours of application, and almost maintained at all over the longer penetration period. The horny layer contains the highest concentrations, with values of 2300–4500 μg/cm³. When CPX-O aqueous cream is spread on the surface of fingernails, the compound is able to penetrate right through the nail. The percutaneous absorption in dogs is higher, at 5–15% of the dose, than that in humans. After vaginal application (1 mg/kg) of 1% CPX-O aqueous cream to bitches, 42–97% of the dose (depending on the animals) is recovered in the urine and feces. The majority (94–98%) of absorbed CPX binds to serum proteins in a concentration range of 0.01–11.0 μg/mL (26). The biological half-life (t_1/2) of CPX is approximately 1.7 hours (26). CPX is excreted by dogs and man in the urine, primarily as a glucuronide (26). The metabolite pattern is similar between oral and dermal applications of CPX. In addition, placental transfer of CPX is low in the rats (26).

Toxicological studies have shown that LD₅₀ (lethal dose 50%) values of CPX in rats and mice are orally 2,500–1,700 mg/kg, subcutaneously 2,500–1,700 mg/kg, intraperitoneally 172–83 mg/kg, and intravenously 79–71 mg/kg (27). Oral administration of CPX does not influence the central nervous system or metabolic functions such as body temperature, urinary and biliary excretion, blood coagulation and acute inflammatory processes (28). Also, oral administration of CPX at 30 mg/kg for 4 weeks or at 10 mg/kg for 3 months does not show any toxic symptoms (e.g. body weight loss and gross organ toxicity), revealing a favorable therapeutic index of CPX (28). However, it has been noticed that oral administration of CPX (> 30 mg/kg/day) over 13 weeks can cause irreversible myocardial degeneration in animals (27). CPX serum concentrations of 10 μM are achievable following repeated administration of the compound to rats and dogs, showing no toxicity (28). Oral administration of CPX (10 mg/kg) does not cause toxicity in healthy human volunteers either (29). Furthermore, according to the document from the US Food and Drug Administration (30), when CPX (1% and 5% solutions in polyethylene glycol 400) is topically applied to female mice twice per week for 50 weeks followed by a 6-month drug-free observation period prior to necropsy, no evidence of tumors is observed at the application sites, indicating that CPX is not carcinogenic. Gene mutation assays in vitro (human A549 cells and BALB/c3T3 cells) and in vivo (Chinese hamster bone marrow) have also demonstrated that CPX is negative in mutagenicity (31). Oral or topical administration of CPX to animals (mice, rats, rabbits, and monkeys) does not exhibit any significant fetal malformations, suggesting that CPX does not cause teratogenicity. Moreover, no reports have shown embryotoxicity or reproductive toxicity in humans (28). However, nursing women are suggested to consult with their doctors before use, since it is not clear whether CPX passes into human milk (6). Less than 5% patients have mild adverse effects, which are generally limited to local rash, itching, and burning, resulting in redness or pain (7). Other adverse effects include headache, erythema, nail disorder, pruritus, alopecia, dry skin, facial edema, and contact dermatitis (27). Taken together, the pharmacological and toxicological profiles indicate that CPX is an effective and safe antifungal agent.

4. ANTICANCER ACTIVITY OF CPX

A study sponsored by Leukemia & Lymphoma Society has identified CPX as the top candidate in a survivin promoter activation screen of 4,800 off-patent drugs and natural products for potential anticancer activity (29). Increasing evidence has demonstrated that CPX possesses anticancer activity against a spectrum of human tumors, including leukemia, lymphoma, myeloma, Ewing’s sarcoma, colon adenocarcinoma, cervical carcinoma, renal cell carcinoma, esophageal cancer, bladder carcinoma, rhabdomyosarcoma, breast carcinoma, and pancreatic ductal adenocarcinoma (Table 1). The anticancer activity of CPX is associated with its inhibiting cell proliferation, inducing apoptosis, suppressing cell migration and invasion, and inhibiting angiogenesis and lymphangiogenesis, as discussed below.

Table 1.

Anticancer Activity of Ciclopirox and Derivatives.

Compound	Effect	In vitro	In vivo	References
CPX-O	Inhibition of RR	Leukemia, myeloma, and solid tumor cells; Primary human acute myeloid leukemia samples	Leukemia xenografts in mice	[29]
CPX	Inhibition of RR	Ewing sarcoma cells	Ewing sarcoma xenografts in mice	[38]
CPX	Blocking eIF5A modification by inhibition of DOHH	Cervical cancer cells		[70]
CPX-O	Inhibition of the eIF5A-hypusine-PEAK1 axis	Pancreatic ductal adenocarcinoma cells (PDAC)	PDAC xenografts in mice	[71]
CPX-O	Inhibition of Wnt/β-catenin signaling	Colon cancer cells	Patients with AML	[76]
CPX-O	Inhibition of CDK4, CDK6, and β-catenin activity	Esophageal cancer cells	Esophageal tumor xenografts in mice	[78]
CPX-O	Inhibition of β-catenin signaling	Myeloma cells		[54]
CPX-O	Inhibition of Wnt/β-catenin signaling	Renal carcinoma cells		[42]
CPX-O	Inhibition of Wnt/β-catenin signaling	Prostate cancer cells		[45]
CPX	Inhibition of KDM4B/Myc	Neuroblastoma cells	Neuroblastoma xenografts in mice	[46]
CPX-O	Inhibition of DOHH and PDH	HUVEC cells		[60]
CPX-O	Inhibition of VEGFR-3/ERK signaling	Lymphatic endothelial cells		[64]
CPX-O	Inhibition of CDKs; downregulation of Bcl-xL and survivin	Rhabdomyosarcoma, breast carcinoma, colon adenocarcinoma cells	MDA-MB-231 xenografts in mice	[34]
CPX-O	Inhibition of CDKs by activating ATR/Chk1 mediated Cdc25A degradation	Rhabdomyosarcoma and breast cancer cells		[37,89]
CPX-O	Inhibition of mTORC1 by activating AMPK, HIF-1/REDD1 and Bnip3 pathways	Rhabdomyosarcoma, breast, prostate, lung, head and neck, skin cancer cells	Rhabdomyosarcoma xenografts in mice	[51,52]
CPX	Inhibition of HPV E6/E7 expression	HPV-positive cervical cancer cells		[93]
CPX-O	Induction of ROS; inhibition of EGFR/Akt	Pancreatic cancer cells	Pancreatic tumor xenografts in mice	[44]
CPX-O	Induction of authophagy by activating ROS-JNK pathway	Rhabdomyosarcoma cells		[41]
CPX-O	Induction of ROS by downregulation of DJ-1	Colorectal cancer cells	Colorectal tumor xenografts in mice	[48]
CPX-O	Induction of ROS and PERK-dependent ER stress	Colorectal cancer cells	Colorectal tumor xenografts in mice	[49]
CPX-O; CPX-POM	Inhibition of Notch, Wnt and Hedgehog signaling	Bladder cancer cells	Mouse BBN model of bladder cancer	[106]

4.1. In Vitro Studies in Cell Cultures

4.1.1. CPX Inhibition of Cell Proliferation

Similar to its fungistatic and fungicidal effects on fungi, CPX displays cytostatic and cytotoxic effects on mammalian cells. Hoffman et al. (1991), for the first time, reported that CPX-O is able to arrest human HL-60 promyeloid leukemia cells at or very near the G₁/S phase boundary (32). More in vitro studies have shown that CPX-O blocks cell cycle progression not only in the G₀/G₁ phase of many tumor cell lines (e.g. human cervical HeLa, breast MDA-MB-231, rhabdomyosarcoma Rh30, neuroblastoma CHP134, and acute lymphoblastic leukemia Jurkat and CEM-C7 cells) (33-37), but also in the S phase of certain tumor cell lines (e.g. human Ewing sarcoma A637 and EW8) (38). Also, the effect of CPX-O on cell cycle progression appears to be concentration-dependent but not time-dependent. This is supported by the observations that high concentrations (6 and 20 μM) of CPX-O effectively blocks neuroblastoma CHP134 cells in the G₀/G₁ phase, while low concentrations (0.6 and 2 μM) of CPX-O leads to a modest accumulation of the cells in the S phase; treatments with 2 or 0.6 μM of CPX-O for 24 and 48 hours display a similar cell cycle distribution (35). CPX-induced cell cycle arrest is attributed to downregulation of cyclins (D1, E, A and B1), cyclin dependent kinases (CDK2, CDK4, and CDK6), cell division cycle 25 A (Cdc25A), and upregulation of CDK inhibitor p21^Cip1 (34, 36, 37). Cell proliferation is closely related to cell cycle progression (39, 40). As a result, CPX is able to inhibit tumor cell proliferation.

4.1.2. CPX Induction of Apoptosis

CPX is cytotoxic to malignant cells. Treatment with CPX-O for 72 hours dose-dependently reduces cell viability of human leukemia (e.g. U937, MDAYD2, NB4, and Jurkat), myeloma (e.g. KMS12, KMS18, THP11, JJN3 and OCI-MY5) and some solid tumor cell lines (colon cancer HCT116 and HT29, and prostate cancer DU145), as well as patient primary leukemic blasts, with LD₅₀ values of ≤ 2.5 μM (29). In contrast, treatment with CPX-O for 72 hours, even at 10 μM, does not reduce the viability of nonmalignant lung fibroblast cell lines (MCR5 and GMO5757) (29), suggesting a tumor-selective cytotoxicity. Similarly, more in vitro studies have shown that CPX-O is able to induce apoptosis in many other solid tumor cells, such as cervical cancer (HeLa) (33), rhabdomyosarcoma (Rh30 and RD) (34, 41), Ewing sarcoma (A637 and EW8) (38), renal carcinoma (A-498 and Caki-2) (42), pancreatic cancer (DanG and PancO2) (43, 44), prostate cancer (LNCaP and BM1604) (45), neuroblastoma (SK-N-AS and SK-N-SH) (46), hepatic carcinoma (Hep3B and HepG2) (47), and colorectal cancer (HCT-8, HCT-8/5-FU, and DLD-1) (48). Mechanistically, CPX chelates intracellular iron, thus inhibiting the iron-dependent enzyme RR, leading to cell death (29). Also, CPX induces apoptosis by downregulation of anti-apoptotic proteins (e.g. Bcl-2, Bcl-xL, Mcl-1, and survivin) (34, 36), induction of protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent ER stress (49), inhibition of Wnt/β-catenin (50), mammalian target of rapamycin (mTOR) (51, 52), and other signaling molecules, as discussed in detail below. As CPX induces protective autophagy, blocking autophagy has been found to enhance CPX-induced apoptosis (41, 48). Furthermore, combination treatment with CPX-O potentiates the cytotoxicity of dexamethasone (36), parthenolide (53), ethacrynic acid (54), and a bi-functional peptide (55) in various tumor cells. These observations indicate that CPX executes its anticancer action partly by inducing apoptosis of cancer cells.

4.1.3. CPX Inhibition of Cell Migration and Invasion

Cancer metastasis is responsible for about 90% of cancer deaths (56). To form metastasis, tumor cells disseminate from the primary tumor, then migrate, invade, settle and grow at a site other than the primary tumor site (56). Apart from its anti-proliferative and pro-apoptotic effects, CPX has anti-migratory and anti-invasive activity as well. It has been shown that CPX is able to inhibit cell motility in a concentration-dependent manner in rhabdomyosarcoma (Rh30 and RD) cells (57). This is partly by suppressing the expression of small GTPases (RhoA, Cdc42 and Rac1) and the phosphorylation of paxillin (57). Also, CPX inhibits the migration of cervical cancer (C33a and SiHa) cells transfected with HPV 16 E6, by blocking E6-induced expression of eIF5A-1 (58). Furthermore, CPX treatment inhibits the migration and invasion of colon cancer (HCT-8, HCT-8/5-FU, and DLD-1) cells (49). This is through altering the expression of proteins related to epithelial-mesenchymal transition (EMT) and invasion, i.e. upregulating E-cadherin and downregulating N-cadherin, Snail, and matrix metallopeptidases (MMP-2 and MMP-9) (49).

4.1.4. CPX Inhibition of Angiogenesis and Lymphangiogenesis

While angiogenesis is essential for the growth of most tumors, lymphangiogenesis is also crucial for metastatic spread (59). Clement et al. (2002) showed that CPX-O inhibits cell proliferation of human umbilical vein endothelial cells (HUVECs) by suppression of deoxyhypusine and proline hydroxylation (60). As a result, CPX-O inhibits angiogenesis as detected by tube-like vessel formation on matrigel and the chick aortic arch sprouting assay (60). In contrast, other studies reported that CPX-O induces hypoxia-inducible factor-1 alpha (HIF-1α) stability, vascular endothelial growth factor (VEGF) expression, and angiogenesis (61-63). Thus, the effect of CPX on angiogenesis is still disputable. In addition, CPX-O has also been found to inhibit the tube formation of lymphatic endothelial cells by inhibiting the expression of vascular endothelial growth factor receptor 3 (VEGFR-3) (64), suggesting inhibition of lymphangiogenesis. All of the above studies were conducted in non-tumor models. Further research is required to determine whether CPX indeed inhibits angiogenesis and lymphangiogenesis in animal tumor models.

4.2. In Vivo Studies in Mouse Xenografts Models

To repurpose CPX for cancer therapy, a number of tumor xenograft mouse models have been used to assess the in vivo anticancer activity of CPX. Eberhard et al. (2009) reported that administration of CPX-O (25 mg/kg) by oral gavage once daily inhibits human leukemia (MDAY-D2, K562 and OCI-AML2) xenograft growth in NOD/SCID mice by up to 65% compared to control treatment without evidence of weight loss or gross organ toxicity (29). Zhou et al. (2010) showed that treatment with CPX-O (25 mg/kg) by oral gavage once daily inhibits human breast (MDA-MB-231) tumor xenograft growth by ~75% in BALB/c nu/nu mice comparing to the treatment with vehicle (control), also showing no weight loss or gross organ toxicity (34). The antitumor effect of CPX-O is attributed to inhibition of cell proliferation and induction of apoptosis, evidenced by Ki-67 and TUNEL staining (34). Similar studies have been performed in human Ewing sarcoma A673 (38) and pancreatic (BxPc3, Panc1 and MIA-PACA-2) tumor xenografts in mice (44). Also, treatment by intraperitoneal injection of CPX (20 mg/kg, once daily) potently inhibits the growth of human colon (HCT-8, HCT-8/5-FU, and DLD-1) tumor xenografts in mice (49). In addition, CPX potentiates the inhibitory effect of gemcitabine on the growth of local pancreatic tumors in BxPC-3 Panc1 and MIA PaCa-2 xenograft models (44). Also, CPX-O enhances the anticancer effect of lenalidomide on murine MPC11 myeloma tumor in BALB/c mice (50). Collectively, these in vivo studies further demonstrate that CPX is a promising anticancer agent.

4.3. Clinical Studies in Patients

The first-in-human phase I study of CPX-O in patients with relapsed or refractory hematologic malignancies (Trial registration ID: NCT00990587) has been completed (65). In this study, CPX-O was found to be stable in the suspension of CPX-O in OraSweet SF® for two weeks under refrigeration and at 25°C with 60% relative humidity. Once-daily dosing of CPX-O at doses of ≤40 mg/m² for five days was well tolerated in all patients tested. However, treatment with CPX-O at 80 mg/m² four times daily caused Grade 3 gastrointestinal dose-limiting toxicities (DLTs) in 3/4 patients. Pharmacokinetic and pharmacodynamic studies showed that orally administered CPX was rapidly absorbed and cleared with a short half-life. The mean maximum serum concentration (C_max) and area under the curve through 6 hours (AUC_(0–6h)) of CPX increased roughly linearly with once-daily dose with a time to C_max (T_max) ranging from 0.5 to 4 hours. After dosing 80 mg/m² once or four times daily, the C_max values exceeded 1 μM. In line with the previous finding (26), the primary route of metabolism for CPX was glucuronidation. The t_1/2 of the drug was less than 6 hours following either single or repeated administration. Suppression of survivin (as a pharmacodynamic marker of biological activity) expression was observed after once-daily CPX-O doses of ≥20 mg/m². Once-daily administration of 40 mg/m² oral CPX-O for five days achieved disease stabilization and/or hematologic improvement in 2/3 patients. The findings highlight that CPX has a great potential to be repositioned for cancer therapy.

5. MECHANISMS OF ANTICANCER ACTION OF CPX

The mechanisms of anticancer action of CPX are multiple and complex. CPX is able to act through chelation of iron (Fe³⁺ and Fe²⁺) (6-8, 21, 27, 29, 52). Current knowledge suggests that most of the anticancer effects of CPX are associated with its iron chelation activity (8). Here we briefly discuss the mechanisms of anticancer action of CPX.

5.1. Inhibition of Ribonucleotide Reductase

Ribonucleotide reductase (RR), which catalyzes the formation of deoxyribonucleotides (DNA precursors) from ribonucleotides, is a key regulator of dNTP biosynthesis in mammals (66) and highly expressed in many cancers (67). RR consists of ribonucleotide reductase M1 (RRM1) and ribonucleotide reductase M2 (RRM2) subunit (66). RRM2 is the iron-dependent subunit (66), so iron chelation by CPX inhibits its function (Fig. 2). It has been demonstrated that CPX-O, as a chelator of intracellular iron, inhibits RR at concentrations that can induce leukemia and myeloma cell death in vitro and in vivo (29). Also, CPX treatment results in a significant reduction in deoxyribonucleotide levels and an accumulation of cells in the S phase due to RRM2 inhibition, causing apoptosis in Ewing sarcoma cells and suppressing the tumor growth in a xenograft mouse model (38). Moreover, the sensitivity of Ewing sarcoma cells to inhibition of RR is enhanced partly by high levels of Schlafen family member 11 (SLFN11), a protein that sensitizes cells to DNA damage (38). These results suggest that CPX is a small molecule inhibitor of RR, which targets the iron center of the RRM2 subunit.

Fig 2.

Ciclopirox inhibits multiple signaling molecules related to cancer development and progression.

5.2. Inhibition of DOHH-eIF5A-PEAK1

Like RR, deoxyhypusine hydroxylase (DOHH) is also an iron-dependent enzyme, which catalyzes deoxyhypusine to hypusine, essential for the maturation and activation of eukaryotic translation initiation factor 5A (eIF5A) (68). Two eIF5A isoforms, eIF5A1 and eIF5A2, participate in the regulation of both initiation and elongation, so they are important for the synthesis of a number of proteins essential for G1/S cell cycle transition (69). It has been described that overexpression of eIF5A in tumor cells is highly correlated with poor prognosis of cancer patients, hence eIF5A1 can serve as a biomarker for malignant growth, and eIF5A2 has been proposed as a candidate oncogene (69). A study has shown that CPX-O is an effective inhibitor of DOHH, thus inhibiting hypusine biosynthesis, leading to cell cycle arrest at G₁ phase (60). Another study has also demonstrated that treatment with CPX results in accumulation of the immature and inactive form of eIF5A containing deoxyhypusine and blocks the proliferation of cervical cancer cells by inhibiting DOHH (70).

Further studies have shown that eIF5A proteins regulate the growth of pancreatic ductal adenocarcinoma (PDAC) cells by modulating the expression of pseudopodium enriched atypical kinase 1 (PEAK1), a non-receptor tyrosine kinase. PEAK1 is reduced and PDAC cell growth is inhibited in vitro and in vivo after CPX treatment or knockdown of eIF5A proteins, suggesting that PEAK1 is one of the targets of eIF5A (71, 72). Collectively, inhibition of the DOHH-eIF5A-PEAK1 axis is one of the mechanisms of anticancer action of CPX (Fig. 2).

5.3. Inhibition of Wnt/β-Catenin

Wnt signaling pathways regulate self-renewal, metabolism, survival, proliferation and epithelial-to-mesenchymal transition (EMT) of target cells (73). Three Wnt signaling pathways have been characterized, including the canonical Wnt/β-catenin pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway (74). Hyperactivation of Wnt/β-catenin signaling links to tumor development and progression due to abnormal cell proliferation and differentiation (75). Hence, inhibition of Wnt/β-catenin signaling results in suppression of tumor growth. Since iron is required for Wnt/β-catenin signaling (76), CPX-O, as an iron chelator, is able to inhibit the Wnt/β-catenin pathway (Fig. 2). It has been shown that CPX-O treatment induces apoptosis and suppresses tumor growth by blocking Wnt/β-catenin signaling in AML patients and murine xenograft models, as well as other tumor cells, including those derived from lymphoma, myeloma, renal, pancreatic, prostate, liver, colorectal, and esophageal cancers (43, 45, 47, 50, 54, 76-79). It appears that CPX-O can promote the degradation of β-catenin in colorectal cancer cells and downregulate the expression of the Wnt target gene axin2 in AML patients (76). Furthermore, inhibition of Wnt with CPX-O and a bi-functional peptide exhibits a synergistic effect against prostate cancer (45). It would be interesting to determine whether CPX affects other components in the Wnt signaling pathways.

5.4. Inhibition of Histone Demethylases/Myc

Histone demethylases promote the activity of oncogenic transcription factors such as Myc, so their activities are associated with poor prognosis of various tumors (80). It is difficult to target Myc directly in anticancer strategies because Myc activity is determined by local chromatin histone methylation status (81). It has been shown that CPX exerts its anticancer action partly by inhibition of several histone demethylases, including KDM4B, which regulates Myc function (81, 82) (Fig. 2). As a result, CPX suppresses the Myc signaling pathway in parallel with mitochondrial oxidative phosphorylation, leading to inhibition of neuroblastoma tumor growth and dissemination in vitro and in vivo. These observations demonstrate that CPX is a novel pan-histone demethylase inhibitor, suppressing Myc activity by epigenetic regulation, which can be exploited for cancer therapy (46).

5.5. Inhibition of Prolyl 4-Hydroxylase

Angiogenesis is crucial for tumor growth and metastasis (59,83). Though the effect of CPX on angiogenesis is controversial (60-63), CPX-O has been found to inhibit prolyl 4-hydroxylase (PHD), a non-heme iron enzyme critical for collagen processing, which leads to inhibition of maturation of collagen in HUVECs, thereby inhibiting angiogenesis (60) (Fig. 2). CPX-O also inhibits the synthesis of DNA and the activity of DOHH, an enzyme essential for hypusine biosynthesis related to eIF4A1 synthesis (9, 60), thus inhibiting cell proliferation of HUVECs (60). The finding is further supported by the report that CPX inhibits Ewing sarcoma growth in part by reducing vasculature development (84).

5.6. Inhibition of VEGFR-3/ERK1/2

Lymphangiogenesis, like angiogenesis, plays an important role in promoting tumor growth and metastasis (85). Vascular endothelial growth factor receptor-3 (VEGFR-3) signaling is crucial for lymphangiogenesis (86). It has been shown that CPX inhibits tube formation in lymphatic endothelial cells (LECs), suggesting inhibition of lymphangiogenesis (64). Mechanistically, this is partly by suppressing VEGFR-3 protein expression and VEGFR-3-mediated phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2) (Fig. 2). Treatment with CPX-O does not alter the mRNA level of VEGFR-3, but inhibits the protein synthesis and promotes the protein degradation of VEGFR-3 (64). More studies are required to determine the underlying mechanisms. Of note, overexpression of VEGFR-3 fails to completely rescue the tube formation inhibited by CPX-O, implying that CPX-O inhibits lymphangiogenesis also involving other mechanisms (64).

5.7. Inhibition of CDKs and Activation of ATR/Chk1

Cyclin-dependent kinases (CDKs) play a key role in the regulation of cell cycle progression, and eventually cell division or cell proliferation (87). The activities of CDKs are precisely regulated by multiple events such as phosphorylation/dephosphorylation of CDKs, as well as bindings of cyclins and CDK inhibitors (87). It has been shown that CPX-O treatment is able to downregulate the protein levels of Cdc25A, cyclins (A, B1, D1 and E), and cyclin-dependent kinases (CDK2, CDK4 and CDK6), and upregulate the protein level of the CDK inhibitor p21^Cip1 (34, 36, 37). Consequently, treatment with CPX-O results in cell cycle arrest in the G₁ phase and inhibition of cell proliferation of a number of tumor cells such as rhabdomyosarcoma, neuroblastoma, acute lymphoblastic leukemia, and breast cancer cells (34, 36, 37) (Fig. 3). Of note, CPX-O has been found to induce cell cycle arrest in the S phase in Ewing sarcoma cells, due to inhibition of RR (38).

Fig 3.

Ciclopirox inhibits CDKs and activates ATR/Chk1, leading to cell cycle arrest at G1 phase.

Cdc25A, a member of Cdc25 family, plays a decisive role in promoting cell cycle progression by removing inhibitory phosphorylation of CDKs (88). Recently we have observed that CPX-O downregulates the protein level of Cdc25A by inducing the phosphorylation of Cdc25A, which promotes the protein degradation of Cdc25A, resulting in increased inhibitory phosphorylation of G1-CDKs and cell cycle arrest at G1 phase in rhabdomyosarcoma (Rh30) and breast cancer (MDA-MB-231) cells (37). Our further research has unveiled that CPX-induced phosphorylation of Cdc25A is attributed to activation of the ataxia telangiectasia and rad3-related (ATR)-checkpoint kinase 1 (Chk1) signaling pathway due to CPX-induced DNA damage in the tumor cells (89) (Fig. 3). Interestingly, addition of ferrous sulfate, but not N-acetyl-L-cysteine (anti-oxidant and ROS scavenger), is able to block CPX-induced DNA double strand breaks and activation of Chk1 (89), suggesting that CPX-induced DNA damage response is due to its iron chelation rather than ROS induction.

5.8. Inhibition of mTORC1

The mammalian target of rapamycin (mTOR) pathway plays a critical role in tumorigenesis and metastasis, so mTOR has become an attractive target in cancer therapy (90, 91). mTOR functions as two complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (90, 91). It has been shown that treatment with CPX-O consistently inhibits mTORC1-mediated phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in various tumor cells (51). CPX-O inhibition of mTORC1 is attributed to its iron chelation, as the inhibitory effect can be blocked by pretreatment with ferrous sulfate (52). Mechanistically, activation of AMP-activated protein kinase (AMPK)-tuberous sclerosis complex (TSC) pathway mainly and activation of both HIF-1/REDD1 (regulated in development and DNA damage responses 1) and Bnip3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3) pathways partially contribute to the iron chelation-induced mTORC1 inhibition (52) (Fig. 4). However, CPX-O may inhibit or activate mTORC2-mediated phosphorylation of Akt in a cell line-dependent manner (51, 52). The mechanism behind the phenomena remains to be determined. Of note, rapamycin, a classical mTORC1 inhibitor, is able to potentiate the anticancer activity of chemotherapeutic agents (92). Interestingly, CPX is also synergistic with various anticancer agents (27, 36, 44, 45, 47, 53, 55).

Fig 4.

Ciclopirox inhibits mTORC1 pathway.

Recently, CPX has also been found to inhibit mTORC1 signaling in human papillomavirus (HPV)-positive cervical cancer cells (HeLa and SiHa), as evidenced by strong inhibition of the phosphorylation levels of both S6K1 and 4E-BP1 (93). However, CPX-induced cellular senescence of these cancer cells is dependent on suppression of HPV E6/E7 oncogene expression rather than inhibition of mTORC1 signaling (93). It appears that CPX inhibits HPV E6/E7 oncogene expression at both RNA and protein levels through chelation of iron, as this inhibitory effect can be counteracted by addition of iron donors (ferrous sulfate and ferric ammonium citrate). Prolonged CPX treatment under normoxic or hypoxic conditions can cause p53-independent activation of caspases-3/7 and apoptosis (93).

5.9. Induction of ROS

ROS can play an important role in tumor development and progression (94, 95). High levels of ROS modulate various cell signaling pathways and alter mitochondrial morphology and potential, triggering intrinsic pathway-dependent apoptosis (95). It has been shown that treatment with CPX-O enhances ROS accumulation in pancreatic cancer cells, which results in inhibition of the epidermal growth factor receptor (EGFR)/Akt signaling, reduction of Bcl-xL and survivin protein levels, and activation of caspase-3, and consequently inhibits cell proliferation and induces cell apoptosis (44) (Fig. 5).

Fig 5.

Ciclopirox induces ROS leading to apoptosis or autophagy.

Growing evidence indicates that ROS can also play a critical role in controlling autophagy (96). Autophagy is an evolutionarily conserved catabolic process that involves the degradation of the components of a cell through the lysosomal machinery (97). Autophagy is a “double-edged sword”. Under some conditions, autophagy contributes to cellular survival by providing nutrients and energy to help cells adapt to starvation or stress (such as hypoxia, irradiation, and anticancer drugs) (98). However, under other settings, activated autophagy leads to autophagic cell death (98). ROS and autophagy are involved in cancer initiation and progression, and both are recognized as the potential targets for cancer treatment (96, 99). It has been shown that CPX-O is able to induce autophagy in human rhabdomyosarcoma cells, which is attributed to induction of ROS-mediated activation of c-Jun N-terminal kinases (JNK) (41) (Fig. 5).

DJ-1 (also known as Parkinson disease protein 7, PARK7) plays a key role in protecting cells from oxidative injury as an endogenous antioxidant (100). CPX-O treatment is able to downregulate the expression of DJ-1 in cervical cancer cells (70). Consistent with this study, treatment with CPX-O cause mitochondrial dysfunction and ROS accumulation by downregulating DJ-1, which is involved in its anticancer activity in colorectal cancer (CRC) cells (48). Meanwhile, DJ-1/ROS axis contributes to activation of AMPK, which provokes CPX-induced autophagy initiation in CRC cells (48) (Fig. 5). It is worth pointing out that CPX-induced autophagy plays a pro-survival role in human rhabdomyosarcoma and CRC cells. Blockage of autophagy markedly enhances the antitumor effect of CPX (41, 48). Whether ROS accumulation results in cell apoptosis or autophagy may depend on environmental cues.

In addition, treatment with CPX-O impairs mitochondrial respiration, promotes aerobic glycolysis, and increases cellular ROS production in CRC cells. As a result, CPX-O treatment can also activate protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent endoplasmic reticulum (ER) stress, provoking caspase-dependent apoptosis (49) (Fig. 5).

6. PATENTS RELATED TO CPX AS AN ANTICANCER AGENT

To reposition CPX for cancer therapy, efforts have been made to synthesize CPX derivatives with high water solubility and bioavailability. Also, a new approach has also been developed for treatment of bladder cancer with CPX and its derivatives.

6.1. Development of CPX-POM Prodrugs

A clinical trial in advanced acute myeloid leukemia (AML) patients has shown that when CPX-O is orally administered as a suspension, its biological activity is not evident at doses of <10 mg/m² (65). This is primarily due to poor oral bioavailability, resulting in low serum concentrations of CPX. Also, the half-life of CPX in the body is less than 6 hours (65). When oral administration of CPX-O at 80 mg/m² four times daily causes gastrointestinal dose-limiting toxicities in patients (65). In addition, the poor water solubility also limits opportunities to deliver CPX via parenteral administration of suitably potent solutions and suspensions. To improve the water solubility and bioavailability, Mehmet Tanol and Scott J. Weir have recently developed CPX prodrugs by adding a phosphoryloxymethyl (POM) or phosphoryloxyalkyl (POA) moiety to CPX through an optimized three-step chemical synthesis process (Table 2) (101-103). In principle, the POM moiety in the CPX-POM prodrugs can be cleaved off by phosphatase enzymes to produce the biologically active drug CPX. As such, when a CPX-POM prodrug is administered to animals or humans, it will be enzymatically cleaved into bioactive CPX within the body rapidly and completely (101-103). This has recently been demonstrated in a preclinical pharmacokinetics study, in which fosciclopirox, a CPX-POM prodrug, has high water solubility and can be formulated to injectable solutions (104). Following intravenous injection, the CPX-POM is rapidly and completely metabolized to its active metabolite, CPX, in rats and dogs, so the bioavailability of the active metabolite is complete. Following subcutaneous administration of fosciclopirox, the systemic bioavailability of CPX is also excellent in rats and dogs. It has been confirmed that the intravenous or subcutaneous bioavailability of CPX-POM is higher than the oral bioavailability of CPX-O in rats and dogs (105). It is expected that intravenous or subcutaneous administration of CPX-POM should have better anticancer efficacy than oral or administration of CPX-O. Currently, a Phase 1 clinical trial (NCT03348514) is recruiting the patients with advanced solid tumors in the US to evaluate the safety, dose tolerance, pharmacokinetics, and pharmacodynamics of intravenous CPX-POM.

Table 2.

Patents for CPX and Derivatives as Anticancer Agents.

Patent Number (Granted Time)	Title	Inventors	References
US8609637B2 (Granted 12/17/2013)	Prodrugs of 6-cyclohexyl-1-hydroxy-4-4methylyridin-2-(1H)-one and derivatives thereof	Mehmet Tanol & Scott J. Weir (University of Kansas)	[101]
JP5853028B2 (Granted 02/09/2016)	Prodrug of 6-cyclohexyl-1-hydroxy-4-methylpyridin-2 (1H) -one and its derivatives	Mehmet Tanol & Scott J. Weir (University of Kansas)	[102]
EP2646035B1 (Granted 03/09/2016)	Prodrugs of 6-cyclohexyl-1-hydroxy-4-4methylyridin2-(1H)-one and derivatives thereof	Mehmet Tanol & Scott J. Weir (University of Kansas)	[103]
US9545413B2 (Granted 01/17/2017)	Methods of forming ciclopirox or derivatives thereof in a subject by administration of prodrug	Mehmet Tanol & Scott Weir (University of Kansas)	[104]
US20170304329A1 (Granted 05/11/2017)	Methods of bladder cancer treatment with ciclopirox, ciclopirox olamine, or a ciclopirox prodrug	Scott J. Weir & Shrikant Anant (University of Kansas)	[105]

6.2. New Methods of Bladder Cancer Treatment with CPX and Its Derivatives

Recently, Scott J. Weir and Shrikant Anant have described new methods of treating bladder cancer with CPX, CPX-O, or a CPX-POM prodrug (called CPX-POM thereafter) (Table 2) (105). On the one hand, CPX, CPX-O, or CPX-POM can be formulated to be suitable for intravenous, subcutaneous, or intramuscular injection, so that the administered drug can be delivered into the kidney, bladder, urethra, or upstream via blood circulation. On the other hand, CPX, CPX-O, or CPX-POM can be formulated with a polymeric carrier, such as nanoparticles, microparticles, or hydrogels, so that the drug can be delivered topically to the bladder cancer cells via bladder instillation. As a result, CPX, CPX-O, or CPX-POM can reach a therapeutically effective amount to inhibit the growth and proliferation of bladder cancer cells (including bladder cancer stem cells) and bladder cancer spheroid formation, through inhibiting ribonucleotide reductase (RR) and disrupting the Notch, TGF-β (transforming growth factor beta), STAT3 (signal transducer and activator of transcription 3), and DNA damage response pathways. Since the CPX-POM has high water solubility and bioavailability (101-104), injection of CPX-POM solution can selectively deliver significant concentrations of active CPX to the entire urinary tract, which overcomes the limitations of topical delivery via bladder instillation. CPX-POM has been investigated for treatment of non-muscle invasive and muscle invasive bladder cancer models in mice (106). Parenteral injection of the CPX-POM (25–200 mg/kg) dose-dependently decreases bladder weight and migration to lower stage tumors in the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model of bladder cancer (106).

7. CONCLUSIONS

Preclinical and clinical studies have demonstrated that CPX, an off-patent antifungal drug, possesses promising anticancer activity in various types of cancer. The anticancer activity of CPX is associated with its induction of cell cycle arrest at G1 phase, suppression of cell proliferation, induction of cell apoptosis, and inhibition of angiogenesis and lymphangiogenesis. It appears that most of the anticancer effects of CPX are attributed to its iron chelation activity. At molecular level, CPX is capable of blocking the activities of certain iron-dependent enzymes, including RR, DOHH, and PHD. CPX, through chelation of iron, can also inhibit the Wnt/β-catenin and mTORC1 pathways, and activates the ATR/Chk1 pathway. Besides, CPX is able to increase the accumulation of ROS by inhibiting the expression of DJ-1. Furthermore, CPX acts as a pan-histone demethylase inhibitor, epigenetically inhibiting the activity of the proto-oncogene Myc. CPX can also suppress the activity of G1-CDKs, and block the VEGFR3/ERK1/2 pathway. However, it is unclear whether CPX impacts all of these signaling molecules or pathways through chelation of iron. Of note, four patents have been issued for the development of CPX prodrugs to improve the water solubility and bioavailability of CPX. One patent has been granted for the development of new methods for treating bladder cancer with CPX, CPX-O, or a CPX prodrug. The pharmacological and toxicological profiles indicate that systemic administration of CPX, especially CPX-POM (CPX prodrug), is feasible and safe. Preclinical and clinical data support that CPX has a great potential to be repositioned for cancer therapy.

List of Abbreviations

4E-BP1

eukaryotic initiation factor 4E binding protein 1

AIDS

acquired immune deficiency syndrome

AML

acute myeloid leukemia

AMPK

AMP-activated protein kinase

ATR

ataxia telangiectasia and rad3-related

BBN

N-butyl-N-(4-hydroxybutyl)-nitrosamine

Bnip3

BCL2/adenovirus E1B 19 kDa protein-interacting protein 3

Cdc25A

cell division cycle 25 A

CDK

cyclin dependent kinase

Chk1

checkpoint kinase 1

CPX

ciclopirox

CPX-G

ciclopirox glucuronide

CPX-O

ciclopirox olamine

CPX-POM

phosphoryl-oxymethyl ester of ciclopirox

CRC

colorectal cancer

DOHH

deoxyhypusine hydroxylase

EGFR

epidermal growth factor receptor

eIF5A

eukaryotic translation initiation factor 5A

EMT

epithelial-to-mesenchymal transition

ER

endoplasmic reticulum

ERK1/2

extracellular signal-regulated protein kinases 1 and 2

HIF-1α

hypoxia-inducible factor-1α

HIV

human immunodeficiency virus

HPV

human papillomavirus

HUVEC

human umbilical vein endothelial cells

JNK

c-Jun N-terminal kinases

LECs

lymphatic endothelial cells

LD50

lethal dose 50%

MCP-1

monocyte chemoattractant protein 1

mTOR

mammalian target of rapamycin

mTORC1

mTOR complex 1

mTORC2

mTOR complex 2

PARK7

Parkinson disease protein 7

PDAC

pancreatic ductal adenocarcinoma

PDH

prolyl 4-hydroxylase

PEAK1

pseudopodium enriched atypical kinase 1

PERK

protein kinase RNA-like endoplasmic reticulum kinase

POA

phosphoryloxyalkyl

POM

phosphoryloxymethyl

SLFN11

Schlafen family member 11

REDD1

regulated in development and DNA damage responses 1

ROS

reaction oxidative species

RR

ribonucleotide reductase

RRM1

ribonucleotide reductase M1

RRM2

ribonucleotide reductase M2

S6K1

p70 S6 kinase 1

STAT3

signal transducer and activator of transcription 3

TGF-β

transforming growth factor beta

TSC

tuberous sclerosis complex

VEGF

vascular endothelial growth factor

VEGFR-3

vascular endothelial growth factor receptor 3