Schistosomiasis is a chronic and debilitating disease, caused by the blood-dwelling flukes of the genus Schistosoma that exacerbates poverty [1,2]. Although close to 800 million individuals are at risk of contracting the disease and over 200 million people are thought to be infected, schistosomiasis is often neglected [3]. The global burden of schistosomiasis has been estimated at 1.7 to 4.5 million disability adjusted life years (DALYs), but even the highest estimate might be an underestimation of the true burden [1,4].

Praziquantel (PZQ) is the only widely used drug for treatment and control of this parasitic infection. Although no clinically relevant resistance to praziquantel has been described to date, development of drug-resistance remains a growing threat, particularly in view of mounting PZQ pressure [5]. Accordingly, in light of emerging resistance against PZQ, and in spite of the challenges associated with developing new antischistosomal drugs, alternatives to PZQ should be identified. There is no dedicated drug discovery and development program pursued for schistosomiasis, either by the pharmaceutical industry or through public-private partnerships. However, despite the paucity of concerted efforts to develop novel antischistosomal drugs, a number of compounds with promising antischistosomal properties have been identified, such as the synthetic trioxolanes [6], the cysteine protease inhibitor K11777 [7], alkylaminoalkanethiosulfuric acids [8], PZQ analogs [9], and the oxadiazoles [10].

Quinine (QN), a quinolone methanol, is the main alkaloid of Cinchona species [11]. Quinine has been used clinically in parenteral treatment of severe malaria and oral treatment of resistant falciparum malaria [12]. Quinine is a fast-acting drug with a short elimination half-life that has been recommended for the treatment of uncomplicated malaria in pregnant women and for drug-resistant malaria for almost 400 years [13]. Quinine is eliminated mainly by hepatic metabolism through cytochrome P450 (CYP) 3A4 with very little (<15%) excreted unchanged in urine [14]. Seven metabolites have been identified with 3-hydroxyquinine (3-HQN) being the major metabolite [15]. Halofantrine (HF), a phenanthrenemethanol derivative of aminoalcohol, was first marketed in 1988. It was used for oral treatment of uncomplicated chloroquine- and multidrug-resistant Plasmodium falciparum malaria in both adults and children [16,17]. Halofantrine is metabolized into N-debutylhalofantrine (HFM) essentially by CYP3A4. Maximal HF concentrations of plasma are reached at 2 µM and its bioavailability is very weak and erratic [17], and hepatic clearance represents a large part of total clearance.

Interestingly, the artemisinins (artemether and artesunate), which are essential components of malaria treatment and control [18], also possess antischistosomal properties [19-21]. Recently, the antischistosomal properties in vitro and in the mouse model of several quinoline antimalarials, of which QN, HF, and mefloquine, with the highest worm reductions observed with mefloquine (>80%) have been reported [22-24]. Meanwhile, Keiser et al. [22] supposed that the relatively lower worm reductions for QN (55%) and HF (51%) and other quinolines could be due to their reduced bioavailability.

Ketoconazole (KTZ), an imidazole derivative, is an orally active, broad-spectrum systemic antifungal agent extensively used in veterinary medicine. Ketoconazole is well known to potently inhibit CYP3A4 activity in human liver microsomes [25]. Since the major metabolic pathway of both QN and HF is through CYP3A4 subfamily [26,27], inhibition of the isoenzyme by KTZ could account for the increased plasma QN and HF levels. In the present study, and in the light of the above-mentioned data, we examined the inhibitory effects of KTZ on the antischistosomal potential of QN and HF against adult S. mansoni infection in mice, by evaluating parasitological, histopathological, and biochemical parameters.

To our knowledge, this is the first experimental investigation documenting the inhibitory effects of the CYP3A4 inhibitor KTZ on the antischistosomal potentials of QN and HF against adult Egyptian S. mansoni infection. In the current work, infection of mice with S. mansoni produced a significant decrease in the hepatic activities of CYP450 and CYT b5 (65.3% and 62.1%, respectively). These findings are in agreement with previous investigations [39-41] and are attributed to denaturation of the CYP450 to its inactive form CYP422 as a result of different pathological reactions that follow egg deposition in the liver, including granulomatous, immunologic, and fibrogenic reactions [42]. Moreover, administration of KTZ in the present investigation before QN or HF enhanced the inhibition in the activities of CYP450 (85.7% and 83.8% vs 45.4% and 52.8%) and CYT b5 (75.5% and 73.5% vs 46.0% and 53.7%, respectively) than observed in mice treated with QN or HF alone. It is likely that this pronounced reduction is the augmenting effect of both KTZ and S. mansoni infection. Previous studies reported that KTZ potently inhibit CYP3A4 activity in human liver microsomes and modifies considerably the pharmacokinetics of CYP3A4 substrates such as tacrolimus [25]. Furthermore, KTZ competitively inhibits CYP3A12, a major isoenzyme of the CYP3A subfamily, and at a therapeutic dose, decreases the total body clearance of the CYP3A substrates midazolam [43] in beagle dogs. In addition, KTZ has inhibitory potency for not only CYP3A but also P-glycoprotein (P-gp) in the rat liver [44]. Likely, our work suggests that oral KTZ affected the hepatic CYP3A4 in mice.

In the present study, the efficacy of QN or HF against adult S. mansoni infection following their administration alone or in addition to KTZ was assessed. Administration of QN or HF alone produced significant reductions in males (59.6% and 54.4%, respectively), females (73.9% and 61.8%, respectively) and total number of worms (65.5% and 57.5%, respectively) compared to infected group. In addition, they decreased hepatic, intestinal tissue egg loads, the percentage of immature and mature eggs and increased the percentage of dead eggs. In this context, Xiao et al. [45] found 80.9-90.3% reduction in total worm burden of adult S. japonicum in mice after a single oral dose of 400 mg/kg of mefloquine, quinine, and quinidine, while halofantrine and lumefantrine showed moderate and poor effects with total worm reductions of 67.5% and 38.4%, respectively. On the other hand, Correa Soares et al. [46] recorded that treatment of S. mansoni-infected female Swiss mice with daily intraperitoneal injections of QN (75 mg/kg/day) from the 11^th to 17^th day after infection caused significant decreases not only in worm burden (39%) but also in female worm burden (40%) and egg production (42%). Moreover, Keiser et al. [22] reported that QN and HF administered orally at a single dose of 400 mg/kg to mice harboring adult S. mansoni (Liberian strain) resulted in total and female worm reductions of 54.7% and 51.7%, and 62.0% and 57.4%, respectively. In the present investigation, it seems that worms of the Egyptian strain of S. mansoni were more susceptible to QN and HF than the Liberian strain.

The detailed mechanism of action of quinoline methanols on schistosomes remains to be investigated. It was demonstrated that the antimalarial chloroquine inhibits the formation of hemozoin (Hz), a heme detoxification aggregate in S. mansoni female homogenates [47]. It is interesting to note that adult female S. mansoni was more affected by QN or HF than male adult S. mansoni. Differences in drug susceptibility between male and female S. mansoni have been reported previously, e.g., following hycanthone treatment [48] and point to a sex-specific interference of the drug with the target, or different drug targets. In addition, it was shown that 4-aminoquinolines interact with "free" heme, hindering its crystallization into Hz. The "free" heme interacts with membranes and exerts severe toxic effects, ultimately killing the parasite through oxidative stress [49]. In this regard, Corrêa Soares et al. [46] reported that interference with heme crystallization in S. mansoni represents an important mechanism by which antimalarial quinoline methanols exerts their antischistosome action. From all of the 3 compounds the authors tested, QN and quinidine (QND) caused the most striking reductions in the Hz content (40-65%) on female worms, which are more susceptible to interference in this process [47]. Hz formation represents the main heme detoxification mechanism in S. mansoni, accounting for more than 50% of total heme content in female worms [50]. Moreover, it was concluded that reduction in total and female worm load, obtained after treatment with QN, QND, and quinacrine (QCR), was positively correlated with the percentage of Hz content reduction in female worms [46]. The present work, together with previous evidence, describes schistosomicidal activity of QN and HF [22,46].

Herein, results revealed that administration of KTZ prior QN or HF significantly enhanced the therapeutic efficacy against S. mansoni infection vs those treated with QN or HF alone. These effects were more pronounced in KTZ+QN-treated group than in KTZ+HF-treated group. In the present study, the enhanced reduction in female and total number of worms in S. mansoni-infected mice treated with KTZ+QN or KTZ+HF was in accordance with the results on the activities of drug-metabolizing enzymes, because mice treated with QN or HF in addition to KTZ showed more inhibition in the activities of CYP450 and CYT b5. Lower metabolism as a result of lower levels of drug-metabolizing enzymes because KTZ possibly leads to increased QN or HF plasma concentrations and to a greater elimination half-life, which in turn increased the length of parasite exposure to the drug.

Schistosome eggs induce liver fibrosis, which is a common pathological process leading to the development of irreversible cirrhosis [51] and inability of the liver to perform its biochemical functions [52]. In the present investigation, the smallest granuloma diameter (50.2% reduction) was recorded in KTZ+QN-treated group followed by QN-treated group (40.5% reduction), KTZ+HF-treated group (40.3% reduction) and PZQ-treated group (32.3%) when compared to the infected group. Moreover, a significant reduction in granuloma diameter was observed in KTZ+QN-treated group vs QN-treated group. Such reductions in granuloma diameter may be attributed to elimination of adult worms by the treatment regimens leading to sustained diminution of egg-induced immunopathology [53] and consequent stopping of further egg deposition [24,54]. The combined treatment regimen of KTZ+QN after S. mansoni infection had an added merit of maximal amelioration of histopathological changes where the liver sections of this group showed remarkably better cellular architecture upon comparing with the S. mansoni-infected untreated and QN-treated groups. Moreover, granuloma in this group showed either partially or almost completely degenerated miracidia beside the minimal surrounding inflammatory reactions. This may be related to either the enhanced killing of the parasite, with marked reduction in egg deposition as evidenced by the parasitological results or because of immunological and anti-inflammatory effects of QN. This is in accordance with Correa Soares et al. [46] who observed that QN treatment promoted remarkable ultrastructural changes in male and female worms, particularly in the gut epithelium and reduced the granulomatous reactions to parasite eggs trapped in the liver. Although nothing is known about the presence or expression of P-glycoprotein (P-gp) in S. mansoni eggs, P-gp has been observed in external layers of the egg shells of the red stomach worm, Haemonchus contortus, a parasitic nematode of ruminants, suggested that P-gp inhibition might contribute to elimination of parasite eggs [55].

Previous studies have reported that schistosomiasis is associated with free radical liberation and disturbance in the cellular antioxidant system [56,57]. The intracellular concentration of reactive oxygen species (ROS) is tightly regulated by multiple defense mechanisms involving ROS scavenging antioxidant enzymes and small antioxidant molecules. In the present study, S. mansoni infection induced a significant decrease in SOD activity, GSH content, serum albumin level, and enhanced lipid peroxidation (malondialdehyde) as well as the serum level of ALT, and these results are in agreement with those previously obtained [20,57-60]. Fernandez-Checa and Kaplowitz [61] reported that this alteration might influence the capability of the liver to provide protection against oxidative damage. The balance between biosynthesis, uptake, oxidation, and export maintains GSH homeostasis at the cellular level. Its reduction in S. mansoni-infected liver tissues is probably related to a reduced synthesis of the tripeptide by the infected liver, which was evidenced by the reduction in serum albumin in this study. It was manifested by increase in malondialdehyde level that lipid peroxidation was enhanced during infection. The decreased activity of SOD in S. mansoni-infected mice may be due to the enhanced lipid peroxidation or accumulation of superoxide radicals and hydrogen peroxide [62].

In the present work, the administration of PZQ alone approximately recovered the contents of GSH, the activities of SOD, and the levels of serum ALT and albumin relative to uninfected group. This could be due to the antischistosomal properties of PZQ as it eradicates parasites, prevents the possible formation of toxic metabolites of S. mansoni worms, abolishes further insults to the liver through interruption of egg deposition, and to its direct effect on mature miracidia within egg granulomas [63] and stopping emission of soluble egg antigens.

Herein, treatment of infected mice with QN or HF alone did not produce any significant change in hepatic antioxidant enzymes or in serum ALT and albumin levels vs those of uninfected group. Similarly, Corrêa Soares et al. [46] reported no changes in malondialdehyde levels in S. mansoni-infected mice treated with QN, which may be attributed to the increased expression of aldehyde dehydrogenase (ADH) gene, which encodes an enzyme involved in detoxification of lipid peroxidation products such as reactive malondialdehyde and hydroxynonenal [64]. On the other hand, Farombi et al. [65,66] reported a decrease in GSH contents and an elevation of malondialdehyde levels upon HF treatment in rat liver microsomes. Moreover, Adaramoye et al. [67] documented that HF treatment significantly reduced GSH and SOD, while increased malondialdehyde in rat liver as well as their serum ALT. Meanwhile, the treatment with either QN or HF alone and in addition to KTZ elevated malondialdehyde levels. Nevertheless, the combined treatment regimens restored serum levels of ALT, albumin, and hepatic GSH (KTZ+HF) to their control values. These results indicate that the liver microsomal lipid peroxidation could be enhanced by quinoline methanols, QN, and HF, and they could have adverse effects on the antioxidant status of the animals. Under such a condition, the need for antioxidants as adjuvant to antimalarials is recommended, and their presence may be crucial to eliminate the products of oxidative reactions.

In conclusion, administration of KTZ, a CYP3A4 inhibitor, with QN or HF produced more inhibition in the activities of CYP450 and CYT b5 that leads to lower metabolism and clearance, which in turn increased the length of parasite exposure to the drug. Accordingly, KTZ enhanced the therapeutic antischistosomal potential of QN and HF over each drug alone. This was evident by increased female, total worm, and egg reductions and healing of hepatic granulomatous lesions, nevertheless, these effects was more obvious in KTZ+QN than in KTZ+HF. QN and HF have adverse effects on the antioxidant status of the animals; therefore, the need for antioxidants as adjuvant to antimalarials is recommended.