Nanoformulations of Albendazole As Effective Anti-Cancer and Anti

Nanoformulations of albendazole as effective anti-cancer and

anti-parasite agents

Abstract

Initially emerging as a widely used clinical anti-parasitic drug, albendazole has been increasingly recognised as an effective anti-cancer agent due to its outstanding advantage, i.e. low toxicity to normal cells but high effectiveness against parasites and some tumours. The major challenge is its poor water solubility and subsequently low bioavailability. This article thus first reviews the brief achievements in using albendazole to treat parasites and cancers, and summarises the basic mechanisms of action of albendazole. Then this article critically reviews recent nanotechnological strategies, i.e. formulating/conjugating it with carriers into nanoformulations, in practices of improving aqueous solubility and efficacy in treatment of tumours and parasites. Our expert opinions in this field are further provided for more effective delivery of albendazole to treat tumours and parasites in vivo.

Keywords: Albendazole nanoformulation, Anti-cancer and anti-parasite, Enhance solubility and bioavailability

1. Introduction

Parasites and cancer cells have many properties in common [1]. Both can live and proliferate unlimitedly in mammals and do not have a regular signalling mechanism. They are able to grow in the absence of exogenous factors, and are resistant to apoptosis with the ability to escape from the immune system and disseminate around the body [2,3]. They are also

1 recognised as having common antigens [4]. It is interesting to note that some anti-cancer drugs, such as imatinib, cisplatin and antifolates, have been used against parasites [2,5-7], and some anti-parasitic drugs, such as manzamine A, suramin, artemisinin and chloroquine, are applicable in cancer therapy [6-9].

Albendazole (ABZ) is an anthelmintic agent from benzimidazole family with a broad spectrum of applications against hydatid cysts and neurocysticercosis [8,9]. Initially, ABZ was used for anti-parasitic effect in 1973 [10], and widely used in clinical chemotherapy for many human cystic echinococcosis or hydatid disease. Based on Australian studies conducted in the

20th century, 47% patients with cysts who were treated with albendazole were considered as cured or improved, while European studies, American studies and other publications reported

76.3%, 53% and 71.5% cure or improvement, respectively [11]. In addition to cystic or hydatid diseases, ABZ is the first choice therapy for microsporidiosis in AIDS, ascariasis, enterobiasis, hookworm infections and neurocysticercosis [12]. According to a WHO report, between 1982, when ABZ was introduced for human use, and 2002 about one billion patients received ABZ manufactured by GlaxoSmithKline (GSK) and a further 200 million received generic products.

Recently, ABZ has attracted attention as a potential anti-cancer agent. The activity against

L1210 mouse leukaemia cells was reported in 1985 [13], and Lacey demonstrated cytotoxicity of ABZ against hepatocellular carcinoma cells (HCC) [14]. Pourgholami et al. tested ABZ further against human SKHEP-1 tumour growth in nude mice and reported profound suppression of tumour growth. Many recent reports have confirmed in vitro and in vivo efficacy of ABZ against a wide range of cancer cells including metastatic melanoma [15], small cell lung cancer [15], colorectal cancer [16], prostate cancer [16] and ovarian cancer [17].

In addition to the efficacy for treatment of these diseases, selective toxicity is a substantial concern in chemotherapy. As is well known, most conventional chemotherapeutic agents affect

2 both cancerous and normal cells, thereby limiting the achievable dose within the tumour and causing side effects [18]. In contrast, recent studies indicate its high killing efficacy to diseased cells, but with low toxicity to normal cells [17,19].

Overall, ABZ is recognised as a safe drug for normal cells with weak toxic effects [20] and is considered promising as an anti-parasitic and anti-cancer drug. However, when it comes to the clinical application, usage is limited due to low solubility in water. Several recent studies have focused on improving ABZ solubility, and the most successful approach is to formulate into nanostructures or nanoformulations. Therefore, we aim to summarize the recent progress in this specific field, including anti-parasite and anti-cancer functions and mechanisms of action of ABZ, and the major efforts to formulate ABZ to host-guest nanocomplexes and nanostructures, which have improved the aqueous solubility and enhanced the activity against parasites and cancers.

2. Anti-parasitic effect of albendazole

Albendazole is considered to be a broad-spectrum anthelmintic drug for treatment of many parasitic diseases. ABZ was initially used just to treat patients who had inoperable conditions, but is now considered a very important part of therapy, either as pure chemotherapy or in combination with surgery [21].

ABZ is highly efficacious in reducing hookworm egg numbers, Giardia antigen and cysts

[22], and is widely used in the treatment of nematodes (roundworms) and parasitic meningitis caused by the nematode Angiostrongylus cantonensis [23]. Eosinophil numbers reduced dramatically and gelatinolytic activity declined. A clinical study in a human model indicated that ABZ treatment led to a remarkable decrease in the number of patients with a severe headache, the most common side effect, while no adverse events were reported [24].

ABZ also plays a major role in the treatment of cestode (e.g. tapeworm) infections [12]. It has been reported that among 253 patients with apparently active Echinococcus granulosus hydatid cysts who were treated with ABZ, 28.5% were regarded as cured, 51% as improved, 18.1% as unchanged and only 2.4% experiencing a worsening of their conditions

[25]. In another study, three patients with liver cysts (two with peritoneal cysts) and five with bone cysts received ABZ treatment for one month and continued repeated treatment after intervals of 2 weeks [26]. The patients with liver cysts were cured and no recurrence in the patients with peritoneal cysts was observed. Very slight improvement in the cases with bone cysts was reported. Moreover, patients with primary multiple cerebral hydatid cysts did not have any cerebral cysts at the end of therapy [27]. Some studies have illustrated that even patients who initially did not show any obvious response may be cured by following up over several years [11].

ABZ is also a key agent for treatment of neurocysticercosis which is caused by the larval stage of Taenia solium [12] and is a serious public health problem in most developing countries

[28]. Administration of ABZ for the treatment of patients with neurocysticercosis for eight days demonstrated effectiveness in 78% cases, while mild, transient gastrointestinal symptoms were observed in 38% of the patients [29,30]. Treating 21 patients with intraparenchymal brain cysticercosis revealed that ABZ was significantly more effective compared with praziquantel in reducing the total number of cysts (88% vs. 50%) [28].

Furthermore, ABZ is an effective therapy for treatment of parasitic fungi. Molina et al. observed rapid and dramatic clinical response and significant reduction of parasite shedding after ABZ treatment of patients with AIDS who had disseminated infection of Septata intestinal

[31]. Dieterich et al. assessed the efficacy of ABZ for the treatment of intestinal microsporidiosis due to Enterocytozoon bieneusi in patients with AIDS [32]. After one month

4 of treatment, an apparent decrease in parasites was observed, with no significant adverse side effect associated with the treatment.

The other anti-parasitic application of ABZ is against chronic strongylodiasis. Treating 52 patients with ABZ led to a 75% cure rate in the first course. Following up with the second course of ABZ treatment for patients who failed in the initial treatment increased the overall cure rate to 81% [33]. Another study indicated a 95% cure rate using ABZ [34]. Efficacy against trongyloides stercoralis was observed after three consecutive days of treatment [35].

In brief, in clinical practice in many developing countries, ABZ is a well-tolerated and fast acting drug with a pivotal role in the treatment of many parasitic diseases, including hydatid disease, neurocysticercosis, chronic strongyloidiasis and microsporidiosis [12].

3. Anti-cancer effect of albendazole

Apart from the high anti-parasite effect, ABZ has been considered as an effective anti-cancer agent in recent years. ABZ has been examined against a wide range of cancer cells including melanoma [15], hepatocellular carcinoma [36], colon [37], colorectal [38], small cell lung cancer [15] and ovarian cancer [19]. Králová et al. showed that treating four intestinal cancer cell lines (i.e. SW480, SW620, HCT8, and Caco2) with ABZ inhibited cell proliferation significantly in a concentration-dependent and time-dependent manner through cell arrest in the G2/M phase [39]. In another study, in vitro ABZ treatment of colorectal cancer cell line

HT-29 and in vivo ABZ treatment of nude mice with peritoneal HT-29 xenografts profoundly inhibited cell growth and peritoneal tumour growth [40]. Further, an ABZ-treated group of

OVCAR-3 tumour-bearing nude mice survived the full length of study while 70% of the control group mice had to be euthanised due to ill health resulting from overt ascites production during the treatment [41].

A recent study showed that ABZ can cause oxidative cleavage on calf-Thymus DNA in

MCF-7 cells, suggesting that this compound can break down DNA [42]. Intracellular ROS levels, oxidative biomarkers and activity of antioxidant enzymes increased with ABZ treatment, and glutathione (GSH) was reduced in animals treated with ABZ, indicating an oxidative stress condition that caused DNA damage and triggered apoptosis signalling [42].

ABZ can also be used in combination with other cancer treatments to improve anti-tumour activity and reduce side effects. Ehteda et al. evaluated the synergic effect of ABZ in combination with paclitaxel, vinblastine, colchicine and 2-methoxyestradiol as the representative drugs [43]. Among the tested drugs, they observed a synergic anti-proliferative effect on HCT-116 cells when ABZ was combined with colchicine or 2-methoxyestradiol.

Consistent with in vitro results, prolonged survival of mice bearing HCT-116 tumours was observed when they were treated with a combination of ABZ and 2-methoxyestradiol.

The synergic effect of ABZ is not limited to chemotherapeutics. Given the fact that ABZ can induce cell cycle arrest at G2/M [44], a radiosensitizing effect of ABZ has been reported in combination with irradiation. Furthermore, the cells co-treated with ABZ and radiation exhibited increased apoptosis after 72 hours [45].

ABZ can also be considered as an effective candidate for suppressing paclitaxel (PTX)- resistant cells. Studying the anti-proliferative effect of ABZ on PTX-resistant ovarian cancer cell lines has revealed that ABZ is highly efficacious in inhibiting proliferation of such cells

[46,47]. Interestingly, compared to non-resistant cells, the paclitaxel-resistant cells elicited higher degrees of sensitivity to the ABZ inhibitory effect [46]. Khalilzadeh et al. assessed the anti-proliferative effect of paclitaxel and ABZ on leukemic cells CEM and the PTX-resistant sub-line CEM/dEpoB300 [48]. Treating CEM and CEM/dEpoB300 with PTX resulted in the

IC50 value of 2.86 and 30.26 nM, respectively, confirming marked resistance of

CEM/dEpoB300 cells to PTX. Remarkably, the IC50 value of ABZ in treating CEM and

CEM/dEpoB300 cells were 0.32 and 0.16 μM, respectively, indicating no resistance of PTX- resistant CEM/dEpoB300 cells to ABZ.

4. Mechanisms of action of albendazole

ABZ can act against parasites and cancer through several mechanisms. Albendazole was reported to inhibit fumarate reductase, which functions as the repiratory chain in many helminths [49] (Figire 1a). In recent years, this drug has been explored as an inhibitor of tubulin polymerization (Figure 1c) and can also arrest G2 and M phase of the cell cycle [50] (Figure

1b). ABZ can also be a potent inhibitor of VEGF and hypoxia inducible factor 1-α, two major factors for parasite and tumour growth, and dissemination [49-51] (Figure 1d). These mechanisms of action of ABZ are schematically illustrated in Figure 1.

4.1. Inhibition of fumarate reductase

Fumarate reductase is an enzyme converting fumarate to succinate. This conversion is important in respiratory chain (anaerobic respiration) of many helminths as well as Krebs cycles in mammalians. It is initially reported that ABZ can inhibit parasites such as ascaris suum [52] and L. donovani and Leishmania [49] by attacking fumarate reductase.

Some recent studies have revealed that fumarate reductase is also active in respiration of some cancerous cells and supports cell survival in nutrition and oxygen deficiency [53].

Furthermore, succinate produced by fumarate reductase from fumarate stabilizes HIF-1 and facilitates angiogenesis for cancer growth [54]. Further studies are required to investigate cancerous cell suppression by ABZ through inhibition of fumarate reductase.

Figure 1. Schematic mechanisms of action of Albendazole. (a). Inhibition of fumarate reductase; (b). arresting cell cycle at G2 and M phase; (c). inhibition of tubulin polymerization; and (d). suppression of VEGF and hypoxia inducible factor 1-α expression.

4.2. Arresting cell cycle at G2 and M phase

ABZ is also anti-proliferative, by inducing cell arrest at the G2/M phase (Figure 1b) in a concentration-dependent and time-dependent manner through cell arrest in the G2/M phase

[39,50]. This anti-proliferation effect has been found in several cancer cells by using ABZ only or in combination with radiation [44,45], leading to a significant increase of cell population at the G2/M phase.

4.3. Inhibition of tubulin polymerisation

Angiogenesis, which is the formation of new blood and lymphatic vessels within the tumour, is a substantial factor in cancer progression [55]. Tubulin binding agents (TBAs) are able to specifically target the cell motility apparatus by affecting the dynamics of microtubules [56].

Microtubules are also a key component of parasites which are vital to the parasites’ ability to invade a host cell [57]. TBAs can bind parasite tubulin and promote disassembly of the parasite microtubular apparatus [52]. This binding leads to mitotic arrest at the metaphase/anaphase transition, followed by cell death induction through apoptosis.

ABZ is well recognised as a tubulin binding agent (TBA) (Figure 1c). ABZ inhibits the polymerisation of the tubulin subunits by selectively binding to β-tubulin through colchicine site, preventing elongation of the microtubules, and depolymerizing subunits [58].

Accordingly, ABZ can bind to the over-expressed tubulin isotype and cause toxicity to parasites or cancer cells, which does not happen to normal cells [14,59].

4.4. Suppression of VEGF and hypoxia inducible factor 1-α expression and ascites formation

Angiogenesis plays a notable role in tumour growth, progression and metastasis, while vascular endothelial growth factor (VEGF) is a potent angiogenic factor and is upregulated in many

9 tumours [60]. By increasing vascular permeability, VEGF can facilitate tumour dissemination via circulation and supply more oxygen and nutrients acting as a survival factor for immature tumours [61]. Correspondingly, many studies demonstrate that tumour growth can be suppressed through inhibiting VEGF [62-64].

Over-expression of VEGF is reported in the presence of many parasites and tumour tissues.

It is found that VEGF is increased in brain tissue and blood of patients with malaria [64,65].

Accumulation of VEGF in parasite-infected red blood cells is also reported [66,67].

Furthermore, innate immune responses triggered by the parasite antigen ultimately lead to the activation of VEGF expression to promote lymph vessel hyperplasia [68]. Recent studies have confirmed that ABZ can profoundly suppress VEGF expression (Figure 1d). Upon treating female nude mice bearing peritoneal tumours of human ovarian cancer cells with ABZ, VEGF expression was significantly inhibited, which was clearly shown in Figure 2 [41]. In another study, downregulation of VEGF type 2 receptor in ROP model of angiogenesis was achieved

[62].

Similar to VEGF, hypoxia inducible factor 1-α (HIF-1α) plays a substantial role in angiogenesis and survival of parasites and tumours. It is reported that anti-VEGF effect of ABZ can be mediated through inhibition of tumoural HIF-1α [51,69].

While ascites formation is a substantial cause of mortality in patients with peritoneal carcinomatosis due to the advanced stage of gastric, colorectal, pancreatic, ovarian and endometrial cancers [41,70], decreasing ascites formation was observed (Figure 3). The decrease of the ascite volume is possibly correlated to the reduction in tumour VEGF level

[41], which may increase the survival rate of the patients.

Figure 2. VEGF levels in plasma (A) and cell-free ascites fluid (B) of vehicle and ABZ–treated mice. In vitro effect of albendazole on VEGF production by incubating SKOV-3 cells for 6 hours with various concentrations of albendazole (0.1-1.0 μmol/L) in culture medium bathing the cells with either albendazole alone (C) or with medium containing albendazole plus 6 ng/mL of MMP-9 as a VEGF stimulator (D) [41].

Figure 3. Cumulative ascites volume (A). The volume of ascites produced per animal per day

(B). A total number of viable OVCAR-3 cells (in million) present in the ascites fluid (C). Effect of albendazole on suppressing the rise in tumour marker level (CA 125 Ku/L; K = 1,000) in the ascites fluid immediately before initiation of drug or vehicle therapy (D) [41].

In another study, the effect of ABZ, paclitaxel and their combination on reducing ascites formation and VEGF mRNA expression was examined [71]. Ascitic fluid accumulation and

VEGF levels were significantly reduced in the three treatment groups compared to the control group, while VEGF mRNA expression was suppressed more significantly in the ABZ-treated group.

Ascites formation is also common for parasites. Many types of parasites can cause liver damage [72-74] and ascitic fluid formation as a result of protein depletion [74], worsening the disease conditions.

In addition to reducing ascites formation caused by cancer, ABZ treatment of patients with eosinophilic ascites for five days led to the disappearance of ascites as well as other signs and symptoms [75]. Another study revealed that ABZ treatment for five days led to no ascitic fluid in the subsequent month [76].

In summary, ABZ is effective in controlling parasitic and cancerous diseases through specifically binding with the microtubulins and inhibiting the expression of VEGF.

5. Strategies for overcoming the limitation of albendazole application for parasite and

cancer treatment

Despite the broad spectrum of applications, ABZ usage is limited due to low water solubility

(2.7 µM) [77], which causes low or variable bioavailability after administration and is considered to be a rate-limiting process in the drug absorption [78]. Many attempts such as using different solvents [79], solid dispersion [80], polymeric dispersion [78], microparticles

[81] and nanoparticles [82] have been investigated to improve the aqueous solubility of ABZ.

Among all, the most successful strategies are formulating albendazole into host-guest nancomplexes and nanoparticles. There are six groups of nanoformulations, including (a). host- guest nanocomplex; (b). Solid-lipid nanocarriers; (c). liposome-like structure; (d). nanocrystal structure; (e). polymer nanostructure; and (f). tiny crystalline dispersed in nanostructured matrix, as schematically outlined in Figure 4.

Figure 4. Albendazole (yellow) nanoformulations in the form of (a). host-guest nanocomplex;

(b). Solid-lipid nanocarrier; (c). liposome-like structure; (d). nanocrystal structure; (e). polymer nanostructure; and (f). tiny crystalline dispersed in nanostructured matrix.

ABZ molecules have been loaded into various nanoparticles, such as albumin nanoparticles

[17], PBCA nanoparticles [83], solid lipid nanoparticles [84], liposomes [85] and copper oxide nanoparticles [86]. Such new ABZ formulations are designed and developed in recent years just to enhance its availability and activity against parasites and cancers. For instance, making albendazole-polybutylcyanoacrylate (PBCA) nanoparticles has improved drug bioavailability from 37% to 76% [87]. The following sections will review the latest progress in relation to host-guest nanocomplexes and nanoparticle formulations of ABZ. As a general summary,

Table 1 lists some examples of ABZ nanoformulations for parasite or cancer treatment for comparison.

Table 1. Nanoformulations of albendazoles for parasite or cancer treatment

ABZ Size Zeta ABZ Referen Composition Application Outcome Advantage Disadvantage nanoformulation (nm) potential dosage ce No significant effect on cell viability, HP-β-CD, citric Ovarian Cancer reduced colony acid/ ascorbic Delayed Cells (1A9, formation by ⁓60%, - - acid/ 1 µM oxidation of [88] OVCAR-3 and no drug toxicity for Sedation and hydrochloric ABZSO Albendazole- SKOV-3) healthy cell line convulsion in acid (pharmacologicall Cyclodextrin (Fresh human animals due to y active Complex hepatocytes) the vehicle metabolite) to Mice bearing toxicity 30% survival after 45 ABZSO2 (the HCT-116 50 days compared with inactive form) - - HP-β-CD, colorectal [89] acetic acid mg/kg no survival after 30 cancer days in control group xenografts Acyclic cucurbit[n]uril- 0.5- Mice bearing Improved, 100% Promising results M1. Albendazole - - type molecular 3.2 SK-OV-3 survival of mice from with a low daily Mild toxicity [77] container mg/kg xenograft 30 to 55 days dose Motor1 Tumour weight Less toxicity in 0.09- Mice bearing reduced from 0.4 g to NP form Efficacy highly Albumin 7-10; THF, 0.1-1% 200- 1.88 SK-OV-3 0.1 g, ascites cells compared to free depends on [17,19] nanoparticles BSA 250 µM xenograft reduced from >200 to form; suppressing particle size 50 million ascites formation Human Safety and Needs Chitosan Tripolyphospha 100 Cell viability reduced 168.8 ± 20mV te, acetic acid/ hepatocellular biocompatibility stabilizing [90] nanoparticles 12.6 μg/mL from 90% to 60% HP-β-CD carcinoma cells of the vehicle agents Large particle More ultrastructural size, no cyst modification in cyst Solid lipid 0.5 Hydatid cyst weight reduction 370 ± Stearic acid, 2.41 mV treated with Low drug dose [91] nanoparticles 140 Poloxamer F68 mg/kg (E.granulosus) compared with albendazole sulfoxide albendazole loaded SLN sulfoxide group 200-fold higher release in NP form compared with free form at pH 7.4 and 100 Ovarian cancer Improved PLGA Poloxamers, 1.5-fold higher High dosage of 260– +27 mV PLGA, chitosan μg/100 cells (Caco-2 stability and [92] nanoparticles 480 release at pH 1.2, cell the drug (as coating) μL cells) mucoadhesion viability reduced from 65% in free form to 30-40% in nanoparticle form Mortality increased from 35% for free albendazole (with/without UV) to Albendazole- Techniques for 60% for ABZ-CuO copper oxide Copper(II) 5-100 Filarial parasite overcoming the 102 -- nitrate, sodium and 85% for ABZ- Cost effective [86] (CuO) μg/mL Setaria cervi limited stability hydroxide CuO with UV, nanocomposites are required higher DNA fragmentation compared to free ABZ

5.1. Albendazole host-guest nanocomplexes

Host-guest nanocomplexes are composed of two or more molecules, and held together by forces other than covalent bonds. Such complexes can increase the solubility and bioavailability of guest molecules. Cyclodextrins (CDs) are cup-shaped molecules with a hydrophilic exterior and a hydrophobic cavity, which together enable this type of molecule to combine with hydrophobic drugs (such as albendazole) to form host-guest nanocomplexes and enhance their solubility [93]. Anionic forms of CDs can form nanospheres or nanocapsules simply via nanoprecipitation techniques [94]. It has been shown that the combination of ABZ and hydroxypropyl-β-cyclodextrin (HPβCD) in a molar ratio of 1/10 resulted in an increase of up to 3,500 times in aqueous solubility [95].

Employing various nanoformulations significantly decreased colony formation (cell integrity and the ability to remain viable and grow into colonies) of OVCAR-3 cells treated with the formulation of ABZ with cyclodextrin (CD) and CD plus citric acid (CA) (Figure 5).

However, no significant difference was caused in viability of cells treated with ABZ-CD, ABZ-

CD+CA ethanolic ABZ or control solution (Figure 5), possibly attributable to the low dose of

ABZ used in all formulations. These results may also indicate that ABZ is active in anti- proliferation by arresting at the G2/M phase, but not so toxic to the cells.

Figure 5. Effect of different ABZ formulations on colony formation (A) and cell viability (B) against OVCAR-3 cells (CD: β-cyclodextrin; CD + CA: β-cyclodextrin plus citric acid) [88].

Another study has also reported a synergic effect between HP-β-CD and citric acid [96].

Using this combination improved the solubility of ABZ 10,000 times compared with that in purified water. Moreover, the pharmacokinetic behavior of ABZ/HP-β-CD/acetic acid revealed that the area under the curve (AUC) of ABZ sulfoxide (ABZSO), the active metabolite of ABZ, was up to 7.3 times higher in mice than that in mice receiving ABZ/hydroxypropyl methyl cellulose (HPMC) formulations [43]. In the subsequent test for in vivo response of HCT-116 xenografts to the ABZ formulations, a much longer survival time (42 days) was observed in the mice group treated with ABZ/HP-β-CD at a low dose (50 mg/kg) than that (32 days) treated with 150 mg/kg ABZ/HPMC as well as that (24 days) of the control mice group (treated with blank vehicle) (Figure 6).

Figure 6. In vivo response of HCT-116 xenografts to ABZ/HP-β-CD and ABZ/HPMC [16]

ABZ/HP-β-CD has also been used to treat T. spiralis. This formulation significantly increased efficacy against adult worms and encysted larvae stages compared with the normal

ABZ suspension [97]. The efficacy of ABZ formulation against encysted worms has great importance since it can be employed to treat inoperable or disseminated cases of systematic helminthic infections while these worms can spread within the body.

Figure 7. M1-ABZ treatment attenuates growth rate of SK-OV-3 tumours. Female, athymic mice with SK-OV-3 xenograft tumours maintained a healthy weight (a,d), attenuated tumour growth rates (b,e) and increased survival (c,f) with once and twice daily dosing of M1·ABZ.

Untreated (closed squares), M1 (closed circles), M1·ABZ once daily (open upright triangle), and M1·ABZ twice daily (open upside down triangle) [77].

Cyclodextrin is also used to synthesise a cross-linked shell micelle that owes its stability to supramolecular forces between β-cyclodextrin and adamantine. The micelles were

demonstrated to have dual functionality: cyclodextrin as a cross-linking point and as a drug

carrier for ABZ, which showed a higher anti-proliferation against human ovarian carcinoma

cell line (OVCAR-3) [98]. Cyclodextrin has also been used in inclusion complex form, which

remarkably improved the physicochemical properties of ABZ and can be considered a suitable

candidate to design oral dosage forms [99].

Some studies have demonstrated that cucurbit[n]uril can also be used as an active host

molecule for insoluble drugs such as ABZ [100,101]. Encapsulating ABZ in cucurbit[6, 7 and

8]uril improved the solubility by 2,000 times [102]. Acyclic cucurbit[n]uril-type molecular

container, Motor1 (M1) has shown potent anti-tumour activity with no sign of side effects

(Figure 7). Here two treatment approaches were used to determine the in vivo efficacy of the

system. One treatment was commenced when tumour volume was approximately 300 mm3

(phase of rapid growth). High doses of M1 (once daily) and M1.ABZ (once and twice daily)

were administrated for 50 days (Figure 7a, 7b and 7c). The other treatment was started when

tumour volume was approximately 100 mm3 (before the phase of rapid growth) and once daily

dosing was administered (Figure 7d, 7e and 7f). The results of both treatments confirmed that

M1.ABZ formulations (1) were non-toxic to healthy cells at high doses (Figure 7a and 7d); (2)

significantly inhibited the growth of SK-OV-3 tumours (Figure 7b and 7e); and (3) significantly

extended the mouse survival (Figure 7c and 7f) [77].

In summary, ABZ host-guest nanocomplexes appear to be a successful strategy to enhance

solubility of ABZ and increase the activity and efficacy for both in vitro and in vivo models.

5.2. Nanoparticles loaded with ABZ

5.2.1. Albumin nanoparticles

Albumin is a biocompatible, biodegradable, non-toxic, and non-immunogenic versatile protein carrier that has a high binding capacity of various drugs [103,104]. Such properties make it an ideal material for fabrication of nanoparticles for the purpose of drug delivery.

Recently, Noorani et al. precipitated ABZ with bovine serum albumin (BSA) to form cross- linked nanoparticles with a size range of 7–10 nm (BSA-ABZ) and 200–250 nm (Nab-ABZ)

[17,19]. In vitro studies demonstrated that Nab-ABZ and free ABZ solution were every effective in inhibiting the proliferation of ovarian cancer cell lines (Figure 8A, 8B and 8C), with Nab-ABZ being slightly more active in treating OVCAR3 and SKOV3 cells. In contrast,

Nab-ABZ was much less effective to the normal ovarian cells than free ABZ (Figure 8D and

8E). Interestingly, the anti-proliferative effect of free ABZ in solution to CHO cells was remarkably reduced through formulating into Nab-ABZ nanoparticle form (Figure 8), demonstrating the advantage of nanoparticle-based formulations.

Figure 8. Comparison of the cytotoxic effects of nab-ABZ vehicle, nab-ABZ 200 nm and free

ABZ in ovarian cancer cells A2780 (A), OVCAR3 (B) and SKOV3(C) and normal ovarian

20 cells HOSE (D) and CHO (E) at different concentrations of ABZ (μM) incubated for 72 hours

[17].

Further examinations revealed that BSA-ABZ 10 nm significantly inhibited cell proliferation at lower concentrations (0.09 and 0.19 µM, Figure 9A and 9B) compared to free

ABZ and Nab-ABZ 200 nm, while no apparent anti-proliferative effect was observed to normal cell lines even at the higher doses (Figure 9C). These results confirm that ABZ has the high anti-proliferative effect on cancer cells, but does not have such an effect to normal cells at these doses. This means that ABZ in BSA-based nanoparticle form is able to kill ovarian cancer cells without affecting the normal ovarian cells. Such a property distinguishes this drug molecule from many chemotherapeutics, most of which affect both cancer and normal cells simultaneously.

Figure 9. Comparison of the cytotoxic effects of BSA-ABZ 10 nm, Nab-ABZ 200 nm and free

ABZ in ovarian cancer cells, OVCAR3 (A) SKOV3 (B) and HOSE (C) at different concentration of ABZ (μM) incubated for 72 hours [105].

Figure 10. Effect of the vehicle, free ABZ, BSA-ABZ 10 nm and Nab-ABZ 200 nm

nanoparticle formulations on BALB/c nude mice bearing OVCAR3 [105].

Noorani et al. also evaluated the anti-tumour efficacy of BSA-ABZ nanoparticle

formulations in vivo and found that intraperitoneal administration of BSA-ABZ 10 nm three

times weekly for three weeks led to a significant reduction of the average tumour weight

compared to free ABZ, whereas the tumour weight was not reduced significantly using Nab-

ABZ 200 nm (Figure 10). In order to confirm relative safety of the system for normal cells,

histological studies seem inevitable. The superior effect of BSA-ABZ 10 nm compared with

Nab-ABZ 200 nm is probably caused by enhanced uptake of nanoparticles due to the size

reduction.

5.2.2. Chitosan nanoparticles

Chitosan is a safe, biodegradable and biocompatible biopolymer that has also been widely examined for ABZ delivery. Through chitosan nanoparticles, the mean extraction recovery for

ABZ in rat plasma was more than 90% [106]. As shown in Figure 11, loading ABZ into chitosan nanoparticles resulted in cellular toxicity superior to or comparable with ABZ combined with HPβCD [107]. Note that the lowest cell viability was observed for cells treated with ABZ dissolved in Tween 20, which seemed to result from use of Tween 20 itself.

Figure 11. Comparison of HepG2 cell viability in blank and albendazole-loaded chitosan

vehicles with common albendazole solvents [107].

In another study, a mixture of Tween 20, chitosan/HPβCD, and tripolyphosphate (TPP) was

used to encapsulate albendazole into chitosan-TPP hybrid nanoparticles, and increased drug

solubility (60 µg/ml) from the hybrid nanoparticles (around 200 nm) was achieved [90]. Liu et

al. used sodium tripolyphosphate as the crosslinking agent and Poloxamer 188 as the auxiliary

solvent to fabricate ABZ-associated chitosan nanoparticles through the emulsion crosslinking

volatile technique [106], and analysed the levels of ABZ and its metabolite ABZ sulfoxide after

administration of this formulation in rat serum. The relative bioavailability of ABZ and ABZ

sulfoxide in rats treated with albendazole-associated chitosan nanoparticle was found to

increase to 146 and 222%, respectively, compared with the rats administered with the ABZ

suspensions. Studies show that various factors such as solution pH, chitosan concentration,

chitosan molecular weight and mass ratio of other reacting agents like sodium tripolyphosphate

can affect the anti-tumour activity of ABZ-chitosan nanoparticles [108]. Chitosan has also been

used to synthesise alginate–chitosan beads as pH-sensitive magnetic targeting system for ABZ

in the gastrointestinal tract [109].

5.2.3. Liposomes

Liposomes are biodegradable, biocompatible, low-toxic nanoparticles with the capability to entrap both hydrophobic and hydrophilic drugs [110], and are thus an alternative carrier for albendazole.

Entrapping ABZ within liposomes has obviously increased its bioavailability via the oral route, as verified by enhanced levels of ABZ and the major metabolites in plasma, liver and cyst compared with the free drug [85]. Furthermore, the increased ABZ bioavailability led to a

75-94% reduction in biomass of the metacestode and a significant increase in survival rate for the animals treated with liposomal ABZ [85].

Liposomal ABZ has also been compared with tablet ABZ [111] in the efficacy of treating

echinococcosis. Significantly, the total effective rate and curative rate using liposomal ABZ

increased to 77.9% and 49.1% from 28.4% and 13.9%, respectively, when tablet ABZ was

used. However, the drug-related adverse effects were very similar (11.1% and 12.7%) for

liposomal and tablet ABZ [111]. In their further clinical study, sixty patients with a single cyst

(CE1) or daughter cyst (CE2) were divided into two groups, which received liposomal and

tablet ABZ treatment, respectively. They found both liposomal and tablet ABZ were very

effective for treating human cysts [112].

5.2.4. Solid lipid nanoparticles

Solid-lipid nanoparticles (SLNs) have been developed as an alternative to traditional colloidal carriers to overcome their limitations. SLNs have the advantages such as improved solubility, possibility of targeted delivery and suitable release profile [84]. For example, SLNs were employed to deliver ABZ through the cyst layers of hydatid cysts [113]. SLNs exhibited a very fast release of ABZ initially, followed by a biphasic release pattern. In vitro studies demonstrated significant enhancement in permeability of ABZ in SLN form into hydatid cysts.

An in vivo study of ABZ sulfoxide and ABZ sulfoxide-loaded SLNs against hydatid cyst revealed that the size and weight of cysts was reduced in the treated animals, although not statistically significantly. In addition, ABZ sulfoxide-loaded SLNs caused more ultrastructural changes to cysts in the treated animals [91].

ABZ-SLNs were also used for treatment of Toxocara Canis infection. Application of pure

ABZ suspension evidently reduced the larvae count in the liver, lung, brain and kidney, but remarkably no larvae were observed in mice treated with ABZ-SLNs [114].

5.2.5. PLGA nanoparticles

Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are considered as successful carriers due

to properties such as biocompatibility, biodegradability, safety (FDA approved) and possibility

of surface modification [10]. PLGA NPs can be used to carry both hydrophilic and hydrophobic

drugs and hence they are good candidates for ABZ delivery. Kang et al. loaded ABZ into PLGA

NPs and further coated NPs with chitosan to improve mucoadhesiveness and colloidal stability

[9]. The concentration of released ABZ in nanoparticle form at physiological pH was 200-fold

higher than free ABZ while it was 1.5-fold higher at pH 1.2. Morphological studies

demonstrated spherical nanoparticles had a higher surface area and were responsible for

enhanced solubility. The system was employed against ovarian cancer cells (Caco-2 cells) and

reduced the cell viability from 65% in free form to 30-40% in nanoparticle form. Moreover,

25 formulating ABZ to chitosan-coated PLGA nanoparticles improved the stability and mucoadhesion.

5.2.6. PBCA nanoparticles

Polybutylcyanoacrylate (PBCA) is a biodegradable polymer which can be made in nanoparticle form in an easier way compared to others [115]. ABZ-loaded PBCA nanoparticles in suspension were colloidally unstable due to aggregation tendency. Stabilisers such as polyvinyl pyrrolidone (PVP), carboxymethylcellulose sodium (CMC-Na) and hydroxy-propyl methyl cellulose (HPMC) were screened for improving stability and fluidity of nanoparticle suspensions [116]. Such an improvement in colloidal stability also enhanced the ABZ intestinal uptake in rats from 21.40% to 43.23% when ABZ-PVP-PBCA nanoparticle suspension was administered to replace the ABZ suspension [117]. A further study showed that ABZ-PVP-

PBCA nanoparticles sustainably released the drug for a much longer time (in vitro).

Furthermore, the uptake of ABZ by rat intestine was improved from 21.40% (in suspension form) to 43.23% using ABZ-PVP-PBCA-NP [118].

5.2.7. Inorganic nanoparticles

To improve anti-filarial effectiveness of ABZ, copper oxide (CuO) nanoparticles were used as an adjuvant to form an ABZ-CuO nanocomposite [86]. Using ABZ-CuO nanocomposite led to higher DNA fragmentation as compared to free ABZ. ABZ is supposed to bind parasitic DNA in an intercalative way and CuO to generate ROS for DNA damage induction, and consequent

DNA fragmentation caused apoptosis of worms [86]. The DNA cleavage studies did not prove any role of ABZ-based copper(II) complexes in cleavage of DNA. Thus DNA cleavage should be relevant to the adjuvant CuO [119].

5.2.8. Pure ABZ nanocrystals

Apart from employing nanocarriers, ABZ can be also made as a pure nanodrug (Figure 4d) in a way similar to other hydrophobic drugs such as hydroxycamptothecin and doxorubicin [120,

121]. Zhang et al. introduced anodised aluminum oxide (AAO) template-assisted method as a versatile and controllable strategy to prepared a pure ABZ nanocrystal formulation [122], which is supposed to increase the solubility of ABZ and bioavailability.

6. Conclusion and Future Perspectives

It is well understood that solubility is a prerequisite to drug absorption, bioavailability and clinical response [123]. Since solubility is a limiting factor in ABZ applications, formulating into nanostructures can be an ideal choice for enhanced ABZ solubility, bioavailability as well as delivery efficacy. As described in Figure 4, there are six types of ABZ nanoformulations developed at the moment in various nanostructures for these purposes. These nanostructured nanoformulations, including host-guest nanocomplexes, liposome, various polymeric and solid lipid nanoparticles, have improved the solubility of ABZ. It is our belief that more suitable nanoformulations are expected and one such a form is very tiny ABZ crystals in a nanostructured biodegradable matrix (Figure 4(f)). This system would enable dual control of the ABZ release, i.e. biodegradation of the matrix to expose ABZ nanocrystals in the first step and then subsequent dissolution of ABZ nanocrystals, which would produce a highly efficient

ABZ formula.

In addition to improved solubility, loading ABZ into nanoparticles has superiority in circulation to ABZ microcrystals or ABZ host-guest molecular form. Another significant property of ABZ nanoformulations is targeted delivery to the tumour tissues via either the enhanced permeability and retention (EPR) effect or ligand-receptor target strategy. The EPR

27 effect leads to leakage of nanoparticles to the tumour tissue through a leaky tumour vasculature and prolonged retention owing to poor lymphatic drainage of tumour [124].

Possibility of target delivery is a vital factor for the safety of ABZ, especially in cancer treatment. It is worth mentioning that a key reason behind the relative safety of ABZ is the low intestinal absorption [125]. When it comes to treatment of parasites, one or few oral doses are necessary; however, in case of cancer treatment, i.v. injections of frequent doses are required.

ABZ nanoformulations can reduce essential doses due to improved bioavailability, but such an improved bioavailability can raise more severe side effects. Although some ABZ nanoformulations did not affect viability and proliferation of healthy cells, further metabolism, histological and behavioural studies are essential for safety confirmation. As a result, nanoparticle-based target delivery is substantial for cancer therapy with ABZ. A promising approach for target delivery is application of stimuli-responsive nanocarriers. For example, diamond nanoparticles are a potential nanocarrier for ABZ with tuneable surface characteristics and high sensitivity to stimuli such as magnetic fields, temperatures, ion concentrations, and spin densities [126, 127].

Many attempts for ABZ nanoformulations have focused on anti-parasite effects and high efficacies in preclinic. By contrast, there are few studies on application of ABZ-loaded nanoparticles (i.e. chitosan and albumin nanoparticles) in preclinical cancer treatment.

Albumin nanoparticles have demonstrated encouraging results as a new strategy in cancer treatment. Obviously, novel nanoparticles, especially hybrid nanoparticles containing ABZ, will need more examinations to confirm applications of ABZ in preclinical cancer treatment.

In addition to developing novel systems for ABZ delivery, there are many opportunities to combine ABZ-loaded nanoparticles with other therapies in both cancer and parasite treatment.

For example, loading ABZ into nanoparticles makes it possible to co-deliver many other therapeutics, such as genes and anti-cancer/anti-parasite drugs. If these drugs are

28 complementary in biofunctions that inhibit the cancer/parasite growth in different ways, the co-delivery will lead to higher efficiency and efficacy.

7. Executive summary

• ABZ is well-recognised as an anti-parasite agent for more than 30 years.

• ABZ has been recognised for anti-cancer effect in recent years.

• One of the outstanding characteristics of ABZ is safe to normal cells with fewer and

weaker side effects.

• ABZ is effective against some drug-resistant cancer cells.

• The clinical efficacy is limited due to low water solubility.

• Nanoparticle-based ABZ delivery systems are promising to improve solubility and

bioavailability.

• Nanoparticle-based ABZ delivery systems may also improve other factors, such as

release pattern and retention time and reduce the risk of side effects.

8. Acknowledgements

The authors acknowledge the financial support from the Australian Research Council (ARC) through Future Fellowship (FT120100813) and Discovery Project (DP170104643), and gratefully acknowledge the University of Queensland Research Scholarship and financial support from the Australian Institute for Bioengineering and Nanotechnology, the University of Queensland.

9. References

1. Ashall F. Cancer cells and parasites: two of a kind. Trends Biochem. Sci. 11(12), 518–

520 (1986).

**2. Dorosti Z, Yousefi M, Sharafi SM, Darani HY. Mutual action of anticancer and

antiparasitic drugs: are there any shared targets? Futur. Oncol. 10(15), 2529–2539

(2014).

This paper discusses the possibility of mutual action of anti-cancer and antiparasitic

drugs, which is important for understanding dual function of albendazole.

3. Klinkert M-, Heussler V. The Use of Anticancer Drugs in Antiparasitic Chemotherapy.

Mini-Reviews Med. Chem. 6(2), 131–143 (2006).

4. Daneshpour S, Bahadoran M, Hejazi SH, Eskandarian AA, Mahmoudzadeh M, Darani

HY. Common antigens between hydatid cyst and cancers. Adv. Biomed. Res. 5, 9

(2016).

5. Gregson A, Plowe C V. Mechanisms of Resistance of Malaria Parasites to Antifolates.

Pharmacol. Rev. 57(1), 117–145 (2005).

6. Guzmán EA, Johnson JD, Linley PA, Gunasekera SE, Wright AE. A novel activity

from an old compound: Manzamine A reduces the metastatic potential of AsPC-1

pancreatic cancer cells and sensitizes them to TRAIL-induced apoptosis. Invest. New

Drugs. 29(5), 777–85 (2011).

7. Sharma N, Thomas S, Golden EB, et al. Inhibition of autophagy and induction of

breast cancer cell death by mefloquine, an antimalarial agent. Cancer Lett. 326(2),

143–154 (2012).

8. Morris DL. Pre-operative albendazole therapy for hydatid cyst. Br. J. Surg. 74(9),

805–806 (1987).

9. Gil-Grande LA, Sánchez-Ruano JJ, García-Hoz F, et al. Randomised controlled trial of

efficacy of albendazole in intra-abdominal hydatid disease. Lancet. 342(8882), 1269–

1272 (1993).

10. Maddison JE, Page SW, Church DB. Small animal clinical pharmacology. .

11. Horton R. Albendazole in treatment of human cystic echinococcosis: 12 years of

experience. Acta Trop. 64(1), 79–93 (1997).

12. Venkatesan P. Albendazole. J. Antimicrob. Chemother. 41, 145–147 (1998).

13. Lacey E, Watson TR. Activity of benzimidazole carbamates against L1210 mouse

leukaemia cells: correlation with in vitro tubulin polymerization assay. Biochem.

Pharmacol. 34(19), 3603–5 (1985).

14. Lacey E. The role of the cytoskeletal protein, tubulin, in the mode of action and

mechanism of drug resistance to benzimidazoles. Int. J. Parasitol. 18(7), 885–936

(1988).

15. Patel K, Doudican NA, Schiff PB, Orlow SJ. Albendazole sensitizes cancer cells to

ionizing radiation. Radiat. Oncol. 6, 160 (2011).

16. Anahid Ehteda, Peter Galettis, Stephanie Wai Ling Chu, Krishna Pilai and DLM.

Complexation of Albendazole with Hydroxypropyl-β-Cyclodextrin Significantly

Improves its Pharmacokinetic Profile, Cell Cytotoxicity and Antitumor Efficacy in

Nude Mice. Anticancer Res. 32(9), 3659–3666 (2012).

17. Noorani L, Pourgholami MH, Liang M, Morris DL, Stenzel M. Albendazole loaded

albumin nanoparticles for ovarian cancer therapy. Eur. J. Nanomedicine. 6(4), 227–

236 (2014).

18. Cho K, Wang X, Nie S, Chen Z, Shin DM. Therapeutic Nanoparticles for Drug

Delivery in Cancer. Clin. Cancer Res. 14(5), 1310–1316 (2008).

**19. Noorani L, Stenzel M, Liang R, Pourgholami MH, Morris DL. Albumin nanoparticles

increase the anticancer efficacy of albendazole in ovarian cancer xenograft model. J.

Nanobiotechnology. 13(1), 25 (2015).

The paper is a good model of an efficient ABZ-loaded nanoparticle for cancer therapy.

20. Gruntman A, Nolen-Walston R, Parry N, Wilborn R, Maxwell H. Presumptive

Albendazole Toxicosis in 12 Alpacas. J. Vet. Intern. Med. 23(4), 945–949 (2009).

21. Horton J. Albendazole for the treatment of echinococcosis. Fundam. Clin. Pharmacol.

17(2), 205–12 (2003).

22. Reynoldson JA, Behnke JM, Gracey M, et al. Efficacy of albendazole against Giardia

and hookworm in a remote Aboriginal community in the north of Western Australia.

Acta Trop. 71(1), 27–44 (1998).

23. Lan KP, Wang ACJ, Lai ASC, et al. The efficacy of therapy with albendazole in mice

with parasitic meningitis caused by Angiostrongylus cantonensis. Parasitol Res. 93(4),

311–7 (2004).

24. Jitpimolmard S, Sawanyawisuth K, Morakote N, et al. Albendazole therapy for

eosinophilic meningitis caused by Angiostrongylus cantonensis. Parasitol. Res.

100(6), 1293–1296 (2007).

25. Horton RJ. Chemotherapy of Echinococcus infection in man with albendazole. Trans.

R. Soc. Trop. Med. Hyg. 83(1), 97–102 (1989).

26. Saimot A, Cremieux AC, Hay JM, et al. Albendazole as a potential treatment for

human hydatidosis. Lancet. 322(8351), 652–656 (1983).

27. Todorov T, Vutova K, Petkov D, Balkanski G. Albendazole treatment of multiple

cerebral hydatid cysts: case report. Trans. R. Soc. Trop. Med. Hyg. 82(1), 150–152

(1988).

28. Takayanagui OM, Jardim E. Therapy for neurocysticercosis. Comparison between

albendazole and praziquantel. Arch. Neurol. 49(3), 290–4 (1992).

29. Cruz I, Cruz ME, Carrasco F, Horton J. Neurocysticercosis: optimal dose treatment

with albendazole. J. Neurol. Sci. 133(1–2), 152–4 (1995).

30. Garcia HH, Gilman RH, Horton J, et al. Albendazole therapy for neurocysticercosis: A

prospective double-blind trial comparing 7 versus 14 days of treatment. Neurology.

48(5), 1421–1427 (1997).

31. Molina J-M, Oksenhendler E, Beauvais B, et al. Disseminated Microsporidiosis Due

To Septata intestinalis In Patients With AIDS: Clinical Features And Response To

Albendazole Therapy. J. Infect. Dis. 171(1), 245–249 (1995).

32. Dieterich DT, Lew EA, Kotler DP, Poles MA, Orenstein JM. Treatment with

Albendazole for Intestinal Disease Due to Enterocytozoon bieneusi in Patients with

AIDS. J. Infect. Dis. 169(1), 178–183 (1994).

33. Archibald LK, Beeching NJ, Gill G V, Bailey JW, Bell DR. Albendazole is effective

treatment for chronic strongyloidiasis. Q. J. Med. 86(3), 191–5 (1993).

34. Pitisuttithum P, Supanaranond W, Chindanond D. A randomized comparative study of

albendazole and thiabendazole in chronic strongyloidiasis. Southeast Asian J. Trop.

Med. Public Health. 26(4), 735–8 (1995).

35. Rossignol JF, Maisonneuve H. Albendazole: placebo-controlled study in 870 patients

with intestinal helminthiasis. Trans. R. Soc. Trop. Med. Hyg. 77(5), 707–711 (1983).

36. Pourgholami M., Woon L, Almajd R, Akhter J, Bowery P, Morris D. In vitro and in

vivo suppression of growth of hepatocellular carcinoma cells by albendazole. .

37. Morris DL, Jourdan J-L, Pourgholami MH. Pilot Study of Albendazole in Patients with

Advanced Malignancy. Oncology. 61(1), 42–46 (2001).

38. Pourgholami MH, Akhter J, Wang L, Lu Y, Morris DL. Antitumor activity of

albendazole against the human colorectal cancer cell line HT-29: in vitro and in a

xenograft model of peritoneal carcinomatosis. Cancer Chemother. Pharmacol. 55(5),

425–432 (2005).

39. Králová V, Hanušová V, Staňková P, Knoppová K, Čáňová K, Skálová L.

Antiproliferative effect of benzimidazole anthelmintics albendazole, ricobendazole,

and flubendazole in intestinal cancer cell lines. Anticancer. Drugs. 24(9), 911–9

(2013).

40. Pourgholami MH, Akhter J, Wang L, Lu Y, Morris DL. Antitumor activity of

albendazole against the human colorectal cancer cell line HT-29: in vitro and in a

xenograft model of peritoneal carcinomatosis. Cancer Chemother. Pharmacol. 55(5),

425–432 (2005).

41. Pourgholami MH, Yan Cai Z, Lu Y, Wang L, Morris DL. Albendazole: a potent

inhibitor of vascular endothelial growth factor and malignant ascites formation in

OVCAR-3 tumor-bearing nude mice. Clin. Cancer Res. 12(6), 1928–35 (2006).

42. Castro LSEW, Kviecinski MR, Ourique F, et al. Albendazole as a promising molecule

for tumor control. Redox Biol. 10, 90–99 (2016).

*43. Ehteda A, Galettis P, Pillai K, Morris DL. Combination of albendazole and 2-

methoxyestradiol significantly improves the survival of HCT-116 tumor-bearing nude

mice. BMC Cancer. 13, 86 (2013).

The paper illustrates possibility of combination therapy with albendazole.

44. Martínez-Espinosa R, Argüello-García R, Saavedra E, Ortega-Pierres G. Albendazole

induces oxidative stress and DNA damage in the parasitic protozoan Giardia

duodenalis. Front. Microbiol. 6, 800 (2015).

45. Patel K, Doudican NA, Schiff PB, Orlow SJ. Albendazole sensitizes cancer cells to

ionizing radiation. Radiat. Oncol. 6, 160 (2011).

*46. Pourgholami MH, Lu Y, Morris DL. Albendazole inhibits paclitaxel-resistant

1A9PTX22 ovarian cancer cells. Cancer Res. 66(8 Supplement), 1271–1271 (2006).

This work shows possibility of employing albendazole against drug-resistant cancer

cells.

47. Chu SWL, Badar S, Morris DL, Pourgholami MH. Potent inhibition of tubulin

polymerisation and proliferation of paclitaxel-resistant 1A9PTX22 human ovarian

cancer cells by albendazole. Anticancer Res. 29(10), 3791–6 (2009).

48. Khalilzadeh A, Wangoo KT, Morris DL, Pourgholami MH. Epothilone-paclitaxel

resistant leukemic cells CEM/dEpoB300 are sensitive to albendazole: Involvement of

apoptotic pathways. Biochem. Pharmacol. 74(3), 407–14 (2007).

49. Chen M, Zhai L, Christensen SB, Theander TG, Kharazmi A. Inhibition of Fumarate

Reductase in Leishmania major and L. donovani by Chalcones. Antimicrob. Agents

Chemother. 45(7), 2023–2029 (2001).

50. Pourgholami MH, Woon L, Almajd R, Akhter J, Bowery P, Morris DL. In vitro and in

vivo suppression of growth of hepatocellular carcinoma cells by albendazole. Cancer

Lett. 165(1), 43–9 (2001).

51. Pourgholami MH, Cai ZY, Badar S, Wangoo K, Poruchynsky MS, Morris DL. Potent

inhibition of tumoral hypoxia-inducible factor 1α by albendazole. BMC Cancer. 10(1),

143 (2010).

52. Barrowman MM, Marriner SE, Bogan JA. The binding and subsequent inhibition of

tubulin polymerization in Ascaris suum (in vitro) by benzimidazole anthelmintics.

Biochem. Pharmacol. 33(19), 3037–3040 (1984).

53. Tomitsuka E, Kita K, Esumi H. The NADH-fumarate reductase system, a novel

mitochondrial energy metabolism, is a new target for anticancer therapy in tumor

microenvironments. Ann. N. Y. Acad. Sci. 1201(1), 44–49 (2010).

54. Selak MA, Armour SM, MacKenzie ED, et al. Succinate links TCA cycle dysfunction

to oncogenesis by inhibiting HIF-? prolyl hydroxylase. Cancer Cell. 7(1), 77–85

(2005).

55. Nishida N, Yano H, Nishida T, Kamura T, Kojiro M. Angiogenesis in cancer. Vasc.

Health Risk Manag. 2(3), 213–9 (2006).

56. Bijman MNA, van Nieuw Amerongen GP, Laurens N, van Hinsbergh VWM, Boven

E. Microtubule-targeting agents inhibit angiogenesis at subtoxic concentrations, a

process associated with inhibition of Rac1 and Cdc42 activity and changes in the

endothelial cytoskeleton. Mol. Cancer Ther. 5(9), 2348–57 (2006).

57. Cottingham K. A new PTM for tubulin is found in a parasite. J. Proteome Res. 9(1), 4–

5 (2010).

58. Akiyoshi DE, Weiss LM, Feng X, et al. Analysis of the b-Tubulin Genes from

Enterocytozoon bieneusi Isolates from a Human and Rhesus Macaque. J Eukaryot

Microbiol. 54(1), 38–41 (2007).

59. Tseng C-Y, Mane JY, Winter P, et al. Quantitative analysis of the effect of tubulin

isotype expression on sensitivity of cancer cell lines to a set of novel colchicine

derivatives. Mol. Cancer. 9(1), 131 (2010).

60. Duffy AM, Bouchier-Hayes DJ, Harmey JH. Vascular Endothelial Growth Factor

(VEGF) and Its Role in Non-Endothelial Cells: Autocrine Signalling by VEGF. In:

Madame Curie Bioscience Database. Landes Bioscience (2013).

61. Moreira IS, Fernandes PA, Ramos MJ. Vascular endothelial growth factor (VEGF)

inhibition--a critical review. Anticancer. Agents Med. Chem. 7(2), 223–45 (2007).

*62. Pourgholami MH, Khachigian LM, Fahmy RG, et al. Albendazole inhibits endothelial

cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and

suppresses retinal neovascularization in ROP model of angiogenesis. Biochem.

Biophys. Res. Commun. 397(4), 729–34 (2010).

The paper investigates important mechanisms of action of albendazole.

63. Heidenreich R, Machein M, Nicolaus A, et al. Inhibition of Solid Tumor Growth by

Gene Transfer of VEGF Receptor-1 Mutants. Int. J. Cancer. 111(3), 348–357 (2004).

64. Deininger MH, Winkler S, Kremsner PG, Meyermann R, Schluesener HJ. Angiogenic

proteins in brains of patients who died with cerebral malaria. J. Neuroimmunol. 142(1–

2), 101–11 (2003).

65. Casals-Pascual C, Idro R, Gicheru N, et al. High levels of erythropoietin are associated

with protection against neurological sequelae in African children with cerebral

malaria. Proc. Natl. Acad. Sci. U. S. A. 105(7), 2634–9 (2008).

66. Hempel C, Hoyer N, Staalsø T, Kurtzhals JA. Effects of the vascular endothelial

growth factor receptor-2 (VEGFR-2) inhibitor SU5416 on in vitro cultures of

Plasmodium falciparum. Malar. J. 13, 201 (2014).

67. Furuta T, Kimura M, Watanabe N. Elevated levels of vascular endothelial growth

factor (VEGF) and soluble vascular endothelial growth factor receptor (VEGFR)-2 in

human malaria. Am. J. Trop. Med. Hyg. 82(1), 136–9 (2010).

68. Pfarr KM, Debrah AY, Spetch S, Hoerauf A. Filariasis and lymphoedema. Parasite

Immunol. 31(11), 664–672 (2009).

69. Aleyasin H, Karuppagounder SS, Kumar A, et al. Antihelminthic benzimidazoles are

novel HIF activators that prevent oxidative neuronal death via binding to tubulin.

Antioxid. Redox Signal. 22(2), 121–34 (2015).

70. Smith E., Jayson G. The Current and Future Management of Malignant Ascites. Clin.

Oncol. 15(2), 59–72 (2003).

71. Choi E-K, Kim S-W, Nam E-J, et al. Differential effect of intraperitoneal albendazole

and paclitaxel on ascites formation and expression of vascular endothelial growth

factor in ovarian cancer cell-bearing athymic nude mice. Reprod. Sci. 18(8), 763–71

(2011).

72. Yagci G, Ustunsoz B, Kaymakcioglu N, et al. Results of Surgical, Laparoscopic, and

Percutaneous Treatment for Hydatid Disease of the Liver: 10 Years Experience with

355 Patients. World J. Surg. 29(12), 1670–1679 (2005).

73. Mahmoud M., El-Abhar H., Saleh S. The effect of Nigella sativa oil against the liver

damage induced by Schistosoma mansoni infection in mice. J. Ethnopharmacol. 79(1),

1–11 (2002).

74. Sangisetty SL, Miner TJ. Malignant ascites: A review of prognostic factors,

pathophysiology and therapeutic measures. World J. Gastrointest. Surg. 4(4), 87–95

(2012).

75. Oncu K, Yazgan Y, Kaplan M, et al. An extremely uncommon case of parasitic

infection presenting as eosinophilic ascites in a young patient. Case Rep.

Gastroenterol. 5(1), 139–43 (2011).

76. Chira O, Badea R, Dumitrascu D, et al. Eosinophilic ascites in a patient with toxocara

canis infection. A case report. Rom. J. Gastroenterol. 14(4), 397–400 (2005).

*77. Hettiarachchi G, Samanta SK, Falcinelli S, et al. Acyclic Cucurbit[n]uril-Type

Molecular Container Enables Systemic Delivery of Effective Doses of Albendazole for

Treatment of SK-OV-3 Xenograft Tumors. Mol. Pharm. 13(3), 809–18 (2016).

The paper is a good model of an efficient ABZ host-guest nanoformulation

78. Leonardi D, Echenique C, Lamas MC, Salomon CJ. High efficacy of albendazole-PEG

6000 in the treatment of Toxocara canis larva migrans infection. J. Antimicrob.

Chemother. 64(2), 375–8 (2009).

79. García A, Leonardi D, Salazar MO, Lamas MC. Modified β-cyclodextrin inclusion

complex to improve the physicochemical properties of albendazole. complete in vitro

evaluation and characterization. PLoS One. 9(2), e88234 (2014).

80. Kalaiselvan R, Mohanta GP, Manna PK, Manavalan R. Studies on mechanism of

enhanced dissolution of albendazole solid dispersions with crystalline carriers. Indian

J Pharm Sci. 68, 599–607 (2006).

81. Barrera MG, Leonardi D, Bolmaro RE, et al. In vivo evaluation of albendazole

microspheres for the treatment of Toxocara canis larva migrans. Eur. J. Pharm.

Biopharm. Off. J. Arbeitsgemeinschaft für Pharm. Verfahrenstechnik e.V. 75(3), 451–

4 (2010).

82. Zafar A, Ahmad I, Ahmad A, Ahmad M. Copper(II) oxide nanoparticles augment

antifilarial activity of Albendazole: In vitro synergistic apoptotic impact against filarial

parasite Setaria cervi. Int. J. Pharm. 501(1–2), 49–64 (2016).

83. Zhang X, Zhang Q, Wen H, Wang G, Sun D. [Preparation of albendazole

polybutycyanocrylate nanoparticles and study on its pharmaceutical properties and

tissue distribution]. Yao Xue Xue Bao. 38(6), 462–6 (2003).

84. Naseri N, Valizadeh H, Zakeri-Milani P. Solid lipid nanoparticles and nanostructured

lipid carriers: structure, preparation and application. Adv. Pharm. Bull. 5(3), 305–13

(2015).

85. Wen H, New RR, Muhmut M, et al. Pharmacology and efficacy of liposome-entrapped

albendazole in experimental secondary alveolar echinococcosis and effect of co-

administration with cimetidine. Parasitology. , 111–21 (1996).

86. Zafar A, Ahmad I, Ahmad A, Ahmad M. Copper(II) oxide nanoparticles augment

antifilarial activity of Albendazole: In vitro synergistic apoptotic impact against filarial

parasite Setaria cervi. Int. J. Pharm. 501(1), 49–64 (2016).

87. Zhang X-N, Tang L-H, Gong J-H, Xu X-Y, Zhang Q. Alternative albendazole

polybutylcyanoacrylate nanoparticles preparation, pharmaceutical properties and tissue

distribution in rats. Lett. Drug Des. Discov. 3(4), 275–280 (2006).

88. Pourgholami MH, Wangoo KT, Morris DL. Albendazole-Cyclodextrin Complex:

Enhanced Cytotoxicity in Ovarian Cancer Cells. Anticancer Res. 28(5A), 2775–9

(2008).

89. Ehteda A, Galettis P, Chu SWL, Pillai K, Morris DL. Complexation of albendazole

with hydroxypropyl-β-cyclodextrin significantly improves its pharmacokinetic profile,

cell cytotoxicity and antitumor efficacy in nude mice. Anticancer Res. 32(9), 3659–66

(2012).

90. Kang B-S, Lee S-E, Ng C, Kim J-K, Park J-S. Exploring the preparation of

albendazole-loaded chitosan-tripolyphosphate nanoparticles. Materials (Basel). 8(2),

486–498 (2015).

91. Ahmadnia S, Moazeni M, Mohammadi-Samani S, Oryan A. In vivo evaluation of the

efficacy of albendazole sulfoxide and albendazole sulfoxide loaded solid lipid

nanoparticles against hydatid cyst. Exp. Parasitol. 135(2), 314–9 (2013).

92. Kang B-S, Choi J-S, Lee S-E, et al. Enhancing the in vitro anticancer activity of

albendazole incorporated into chitosan-coated PLGA nanoparticles. Carbohydr.

Polym. 159, 39–47 (2017).

93. Ping Y, Liu C, Zhang Z, Liu KL, Chen J, Li J. Chitosan-graft-(PEI-β-cyclodextrin)

copolymers and their supramolecular PEGylation for DNA and siRNA delivery.

Biomaterials. 32(32), 8328–8341 (2011).

94. Lakkakula JR, Maçedo Krause RW. A vision for cyclodextrin nanoparticles in drug

delivery systems and pharmaceutical applications. Nanomedicine. 9(6), 877–894

(2014).

95. Bassani VL, Krieger D, Duchene D, Wouessidjewe D. Enhanced water-solubility of

albendazole by hydroxypropyl-?-cyclodextrin complexation. J. Incl. Phenom. Mol.

Recognit. Chem. 25(1–3), 149–152 (1996).

96. Evrard B, Chiap P, DeTullio P, et al. Oral bioavailability in sheep of albendazole from

a suspension and from a solution containing hydroxypropyl-β-cyclodextrin. J. Control.

Release. 85(1), 45–50 (2002).

97. Garcı́a JJ, Bolás F, Torrado JJ. Bioavailability and efficacy characteristics of two

different oral liquid formulations of albendazole. Int. J. Pharm. 250(2), 351–358

(2003).

98. Yhaya F, Binauld S, Kim Y, Stenzel MH. Shell cross-linking of cyclodextrin-based

micelles via supramolecular chemistry for the delivery of drugs. Macromol. Rapid

Commun. 33(21), 1868–74 (2012).

99. García A, Leonardi D, Salazar MO, et al. Modified β-Cyclodextrin Inclusion Complex

to Improve the Physicochemical Properties of Albendazole. Complete In Vitro

Evaluation and Characterization. PLoS One. 9(2), e88234 (2014).

100. Saleh N, Khaleel A, Al-Dmour H, al-Hindawi B, Yakushenko E. Host–guest

complexes of cucurbit[7]uril with albendazole in solid state. J. Therm. Anal. Calorim.

111(1), 385–392 (2013).

101. Nora M, Fatiha M, Leila N, Sakina H, DjamelEddine K. Density functional study of

inclusion complex of Albendazole/cucurbit [7]uril: Structure, electronic properties,

NBO, GIAO and TD-DFT analysis. J. Mol. Liq. 211, 40–47 (2015).

102. Zhao Y, Buck DP, Morris DL, Pourgholami MH, Day AI, Collins JG. Solubilisation

and cytotoxicity of albendazole encapsulated in cucurbit[n]uril. Org. Biomol. Chem.

6(24), 4509–15 (2008).

103. Yu Z, Yu M, Zhang Z, Hong G, Xiong Q. Bovine serum albumin nanoparticles as

controlled release carrier for local drug delivery to the inner ear. Nanoscale Res. Lett.

9(1), 343 (2014).

104. Elzoghby AO, Samy WM, Elgindy NA. Albumin-based nanoparticles as potential

controlled release drug delivery systems. J. Control. Release. 157(2), 168–182 (2012).

105. Noorani L, Stenzel M, Liang R, Pourgholami MH, Morris DL. Albumin nanoparticles

increase the anticancer efficacy of albendazole in ovarian cancer xenograft model. J.

Nanobiotechnology. 13(1), 25 (2015).

106. Liu Y, Wang X, Ren W, et al. Novel albendazole-chitosan nanoparticles for intestinal

absorption enhancement and hepatic targeting improvement in rats. J. Biomed. Mater.

Res. B. Appl. Biomater. 101(6), 998–1005 (2013).

107. Kang B-S, Lee S-E, Ng CL, Cho C-W, Park J-S. Determination of preparation

parameters for albendazole-loaded nanoparticles using chitosan and tripolyphosphate.

J. Pharm. Investig. 45(3), 265–269 (2015).

108. Torabi N, Dobakhti F, Haniloo A. Albendazole and Praziquantel Chitosan

Nanoparticles: Preparation, Characterization, and In Vitro Release Study. Iran. J. Sci.

Technol. Trans. A Sci. , 1–7 (2017).

109. Wang F-Q, Li P, Zhang J-P, Wang A-Q, Wei Q. A novel pH-sensitive magnetic

alginate–chitosan beads for albendazole delivery. Drug Dev. Ind. Pharm. 36(7), 867–

877 (2010).

110. Anabousi S, Laue M, Lehr C-M, Bakowsky U, Ehrhardt C. Assessing transferrin

modification of liposomes by atomic force microscopy and transmission electron

microscopy. Eur. J. Pharm. Biopharm. 60(2), 295–303 (2005).

111. Li H, Shan J, Shao Y, et al. Clinical effect and safety of liposomal-albendazole and

tablet-albendazole for the treatment of human cystic echinococcosis. Chinese J.

Hepatol. 19(7), 532–6 (2011).

112. Li H, Song T, Shao Y, Aili T, Ahan A, Wen H. Comparative evaluation of liposomal

albendazole and tablet-albendazole against hepatic cystic echinococcosis. Medicine

(Baltimore). 95(4), e2237 (2016).

113. Soltani S, Rafiei A, Ramezani Z, Abbaspour MR, Jelowdar A, Sagha Kahvaz M.

Evaluation of the hydatid cyst membrane permeability of albendazole and albendazole

sulfoxide-loaded solid lipid nanoparticles. Jundishapur J. Nat. Pharm. Prod.

Inpress(Inpress) (2016).

114. Kudtarkar A, Shinde U, Bharkad G, Singh K. Solid Lipid Nanoparticles of

Albendazole for Treatment of Toxocara Canis Infection: In-Vivo Efficacy Studies |

BenthamScience. Nanosci. Nanotechnology-Asia . 6 (2016).

115. He W, Jiang X, Zhang Z-R. Preparation and Evaluation of Poly-Butylcyanoacrylate

Nanoparticles for Oral Delivery of Thymopentin. J. Pharm. Sci. 97(6), 2250–2259

(2008).

116. Xue-Nong Z, Jing-Hong G, Li-Hua T, Qiang Z. Studies on the Drug-Loading

Mechanism of Polybutylcyanocrylate Nanoparticle and its Stability of

Thermodynamics. Curr. Nanosci. 4(1), 59–61 (2008).

117. Zhang XN, Chen J, Zhang Q. Study on the release of albendazole nanoparticles in

vitro and its correlation with uptake in stomach and intestinal absorption kinetics in

rats. Chinese Pharm. J. 38(12), 932–935 (2003).

118. Zhang XN, Zhang Q, Wen H, Wang GQ, Sun DJ. Preparation of albendazole

polybutycyanocrylate nanoparticles and study on its pharmaceutical properties and

tissue distribution. Yaoxue Xuebao. 38(6), 462–466 (2003).

119. Ibrahim MM, Ramadan A-MM, El-Sheshtawy HS, Mohamed MA, Soliman M, I.M.

Zayed S. Synthesis, characterization and medical efficacy (hepatoprotective and

antioxidative) of albendazole-based copper(II) complexes – an experimental and

theoretical approach. J. Coord. Chem. 68(24), 4296–4313 (2015).

120. Zhang R, Li Y, Cai Q, et al. Preclinical pharmacology of the natural product anticancer

agent 10-hydroxycamptothecin, an inhibitor of topoisomerase I. Cancer Chemother.

Pharmacol. 41(4), 257–267 (1998).

121. Yu C, Zhou M, Zhang X, et al. Smart doxorubicin nanoparticles with high drug

payload for enhanced chemotherapy against drug resistance and cancer diagnosis.

Nanoscale. 7(13), 5683–5690 (2015).

**122. Zhang J, Li Y, An F-F, Zhang X, Chen X, Lee C-S. Preparation and Size Control of

Sub-100 nm Pure Nanodrugs. Nano Lett. 15(1), 313–318 (2015).

This paper presents a good approach for fabricating a nanodrug formulation for

hydrophobic drugs, which is applicable for alobendazole nanoformulation.

123. Amidon GL, Lennernäs H, Shah VP, Crison JR. A Theoretical Basis for a

Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product

Dissolution and in Vivo Bioavailability. Pharm. Res. 12(3), 413–420 (1995).

124. Kobayashi H, Watanabe R, Choyke PL. Improving conventional enhanced

permeability and retention (EPR) effects; what is the appropriate target? Theranostics.

4(1), 81–9 (2013).

125. JUNG H, MEDINA L, GARCÍA L, FUENTES I, MORENO-ESPARZA R.

Biopharmaceutics: Absorption Studies of Albendazole and Some Physicochemical

Properties of the Drug and Its Metabolite Albendazole Sulphoxide. J. Pharm.

Pharmacol. 50(1), 43–48 (1998).

126. Chen X, Zhang W, Wang K, et al. Diamond nanostructures for drug delivery,

bioimaging, and biosensing. Chem. Soc. Rev. 46(3), 734–760 (2017).

127. Kaur R, Badea I. Nanodiamonds as novel nanomaterials for biomedical applications:

drug delivery and imaging systems. Int. J. Nanomedicine. 8, 203–20 (2013).

Pourgholami MH1, Woon L, Almajd R, Akhter J, Bowery P, Morris DL. In vitro and in vivo suppression of growth of hepatocellular carcinoma cells by albendazole, Cancer Lett. 165, 43-9 (2001)