[beta]-Lapachone: a naphthoquinone with promising antischistosomal properties in mice.
The activity of p-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione, [beta]-lap) against different stages of Schistosoma mansoni was investigated in mice. Mice infected with 50 cercariae (BH strain) were intraperitoneally treated at a dose of 50 mg/kg for 5 consecutive days, starting on the 1st, 14th, 28th and 45th days after infection, to evaluate the effect of [beta]-lap on skin schistosomula, lung schistosomula, young worms (before oviposition) and adult worms (after oviposition), respectively. All animals were euthanized 60 days after infection. [beta]-Lap significantly reduced (p < 0.001) the number of worms in 29.78%, 37.2%, 24.2% and 40.22% when administered during the phases of skin schistosomula, lung schistosomula, young worms and adult worms, respectively. Significant reduction was also achieved in terms of female burden. In all groups, there was significant reduction in the number of eggs and granulomas in the hepatic tissue. When the intervention was performed during the phase of adult worms, [beta]-lap reduced the size of hepatic granulomas and changed the oogram pattern, lowering the percentage of immature eggs and increasing the percentage of mature and dead eggs. Our data indicate that [beta]-lap has moderate antischistosoma] properties. Its molecule may also be used as a prototype for synthesis of new naphthoquinone derivatives with potential schistosomicidal properties. Further studies with different formulations containing [beta]-lap are needed to clearly establish the best dose and route of administration and its mechanism of action against schistosomes.
Schistosomiasis is a chronic and debilitating disease caused by worms of the genus Schistosoma. The infection is potentially life-threatening with severe pathological manifestations in the hepato-splenic system and gastrointestinal tract (Neghina et al., 2009). It is included on the World Health Organization (WHO)'s list of neglected diseases and has a significant economic and social impact. It is estimated that 779 million people are at constant risk of infection and 207 million are infected, of whom 120 million are symptomatic, 20 million develop the chronic form and more than 500,000 die each year (Steinmann et al., 2006).
Chemotherapy is the only immediate measure for reducing the incidence and prevalence of schistosomiasis in endemic areas, since, to date, there is no satisfactory vaccine against the parasite. At present, praziquantel (PZQ) is the only drug recommended for the treatment and control of schistosomiasis, although it is not active against schistosomula and young worms and does not prevent reinfection (Sabah et al., 1986; Doenhoff et al., 2000). More than 100 million people are currently being treated with PZQ. The huge demand for its use is confirmed not only by the high prevalence of schistosomiasis, but by countless cases of reinfection which are portrayed in an annual or semiannual basis. This leads to the development of resistant parasites and consequently to a collapse in the treatment of the disease. This has encouraged us to contribute with new studies to preserve PZQ in the treatment of schistosomiasis.
[beta]-Lapachone [beta],4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione, P-lap, Fig. 1) is a bioactive quinone, originally isolated from the heartwood of trees of the Bignoniaceae family (Tabebuia sp.). It can also be found in other families such as Verbenaceae, Proteaceae, Leguminosae, Sapotaceae, Scrophulariaceae and Malvaceae. The great interest in its study, including the elucidation of mechanisms of action of compounds with quinoidal structure is due to their multiple biological targets and their widespread presence in nature (Hussain et al., 2007).
[beta]-Lap is active against bacteria (Lourenfo et al., 2011; Kenneth et al., 2012; Silva et al., 2009), viruses (Schuerch and Wehrli, 1978; Li et al., 1993), fungus (Medeiros et al., 2010) and the protozoa Plasmodium (Andrade-Neto et al., 2004; Perez-Sacau et al., 2005) and Trypanosoma (Menna-Barreto et al., 2007; Salas et al., 2008; Ferreira et al., 2011). In addition to these effects, other bioactivities of [beta]-lap have been reported, such as the anti-inflammatory activity (Sitonio et al., 2012) and especially the powerful anticancer activity, displaying selective activity against various human cancers (Bey et al., 2007; Moon et al., 2010).
The aim of this study was to describe the effect of P-lap in the developmental stages of Schistosoma mansoni, based on parasitological and histopathological criteria in a model using mice. In a bibliographical survey, we found no studies on in vivo antischistosomal activities of [beta]-lap, these activities being described here for the first time.
Materials and methods
Female Swiss albino mice, 30-day old, weight ~30g, were obtained and kept in the Laboratory of Immunopathology Keizo Asami pound, in a controlled environment (temperature between 20[+ or -] 2[degrees]C, 12 h daylight cycle), having free access to food (Labina[R]/Purina, Sao Paulo, SP) and water ad libitum. All animal experiments were conducted after the approval from the Animal Experiments Ethics Committee of the UFPE Protocol number 23076.020127/2010-47.
Infection of mice
The mice were infected with the BH strain of S. mansoni maintained at the Laboratory of Immunopathology Keizo Asami of the Federal University of Pernambuco (UFPE - Recife, PE, Brazil), through successive passages in Biomphalaria glabrata and Swiss mice. Cercariae from at least 80 shedding B. glabrata snails were used to infect the mice. Each mouse was percutaneously infected with 50 cercariae (Olivier and Stirewalt 1952).
[beta]-lap was synthesized in the Department of Antibiotics of the UFPE from lapachol extracted from the sawdust Tabebuia avellanedae, according to the methodology described by Lima et al. (1962). [beta]-lap was solubilized in 10% dimethyl sulfoxide (DMSO), 1% Tween 80 in saline solution. Praziquantel (PZQ) (Sigma Chemical, St. Louis, MO, USA) was suspended in 2% Cremophor EL in saline solution.
While planning the experimental design two criteria were taken into account: (1) the onset of [beta]-lap administration and (2) the dose. The time of administration was used to take into account the schistosome life cycle stages, because the time of exposure of schistosomes to a drug may often be of greater importance than the dose applied (Gonnert and Andrews, 1977). The mice were randomly allocated into four experiment groups (ten mice each) according to initiation of treatment. Treatment was started on the 1st, 14th, 28th and 45th day after infection to evaluate the following stages of development: skin schistosomula, lung schistosomula, young worms (stages before oviposition) and adult worms (stage after oviposition) (Fig. 2).
[beta]-lap was injected intraperitoneally at a dose of 50 mg/kg of body weight/day. The injections were administered during five consecutive days and freshly prepared before administration. The dose used was based on experiments that describe the activities of [beta]-lap against other parasites (Andrade-Neto et al., 2004).
Each experimental group treated with the [beta]-lap was accompanied by an untreated infected control group (ten mice each) that received the drug vehicle only. A positive control group was established to receive the reference drug PZQ, administered in the dose of 50 mg/kg/day, orally through gavage, from the 45th until the 49th day after infection. This guideline ensures high parasitological cure rate within the period in which the infection presents adult worms, but there is no indication in periods in which the infection presents immature worms. For these stages, there is no drug available to treat schistosomiasis (Sabah et al., 1986; Doenhoff et al., 2000).
All animals were euthanized by cervical dislocation 60 days after infection.
Recovery of worms
The worms were recovered from the hepatic portal system and mesenteric vessels through the technique described by Smithers and Terry (1965). The percentage reduction in the number of worms after treatment was calculated as follows: % reduction = C - V/C x 100, where C=the mean number of parasites recovered from infected untreated animals and V=the mean number of parasites recovered from treated animals.
Fragments from different sites in the small intestine were removed and used to evaluate the development and maturation of the S. mansoni eggs, as described by Pellegrino et al. (1962).
Ova count in hepatic tissue
To estimate the number of eggs per gram of hepatic tissue, a sample of the liver with about 0.3 g was removed from the central remaining part of the right lobe of each animal and processed separately using the potassium hydroxide (KOH) digestion technique (Cheever, 1968).
Hepatic histopathology and granuloma measurement
For each perfused mouse, a sample of the liver was removed from the central part of the left lateral lobe and fixed in 10% buffered formaldehyde and embedded in paraffin blocks to obtain thin histological sections (5 p,m) that were stained with hematoxylin and eosin. Ten random fields per histological sample were used to count the average number of granulomas which were evaluated histologically using light microscopy. Twenty granulomas of each animal containing a single central egg were randomly selected and used to determine the average diameter of granulomas by measuring two perpendicular diameters transmiracidial at the average level of the egg (Phillips et al., 1977). Images were obtained by using an optical microscope connected to a digital camera and a computer system (Motic Images Plus 2.0 ML[TM]). All analyses were performed by double-blind observers.
Statistical analysis was performed using GraphPad Prism 3.02. Data were expressed as the mean [+ or -] SD. All variables were compared using Student's t-test. In all cases, results were considered significant at p < 0.05.
Effect of [beta]-lap on different development stages of S. mansoni
Among the control groups used in the study, there were no significant differences in the total number of worms (overall average, 22.79 [+ or -] 2.93; p = 0.64) and female worms (overall average, 10.09; p = 0.91), showing uniformity on the infection.
The administration of [beta]-lap in mice harboring different development stages of S. mansoni--skin schistosomule (1 day after infection), lung schistosomula (14 days after infection), young worms (28 days after infection) and adult worms (45 days after infection), significantly reduced (p< 0.001) the overall worm burden by 29.78%, 37.2%, 24.2% and 40.22% respectively, when compared to the control group. A similar percentage reduction was also achieved in the recovery of female worms for the same schemes of therapeutic intervention. When analyzing the number of eggs per gram of hepatic tissue, [beta]-lap induced significant reductions (p< 0.001) of 32.71%, 42.07%, 34.64% and 48.01% were achieved when the drug was administered on the 1st, 14th, 28th and 45th day after infection, respectively. The PZQ dose used, in 45-day-infected mice, showed high percentage in reducing the overall rate of worm burden (94.66%) and worm burden females (93.66%) and a reduction of 65.78% in the hepatic tissue egg load (Table 1).
In all groups treated with 3-lap, intestinal tissue samples exhibited S. mansoni eggs at all development stages as was seen in the control groups (data not shown). However, during the adult worms stage, compared to control, [beta]-lap and PZQ significantly reduced the percentage of immature eggs in 20% and reaching zero percent, respectively. Even at this stage, both [beta]-lap as PZQ significantly increased the percentage of dead eggs, however this effect was more evident with PZQ. Regarding mature eggs [beta]-lap increased in 13.2% while PZQ reduced in 25.18% (Fig. 3).
Effect of [beta]-lap on hepatic schistosomotic granuloma
The hepatic lobular architecture was found to be preserved in both control and treated mice. Histopathological examination of the hepatic tissue sections in control groups exhibited typical inflammatory granulomatous reaction in the hepatic parenchyma and portal tract with an infiltration of eosinophils, polymorphonuclear neutrophils, in addition to a few macrophages and lymphocytes surrounding the egg (Fig. 4A and D). This profile of inflammatory reaction was also seen in the groups treated with [beta]-lap during immature stages of the worm. When animals were treated with PZQ or [beta]-lap, 45 days after infection, the exudative granulomatous reaction showed less eosinophilic infiltration, granuloma circumscription and eggs degeneration more evidently than in untreated infected controls (Fig. 4C and E).
There was significant reduction (p < 0.0001) in the average number of hepatic granulomas in all the groups treated with [beta]-lap or PZQ (Table 1). Fig. 4A and B shows the difference in the numerical density of periovular granuloma distributed in the hepatic tissue of untreated-infected mice and mice treated with 3-lap 45 days after infection, respectively.
Only the group treated with the [beta]-lap 45 days after infection showed significant reduction of 25.8% in the average diameter of the granuloma when compared to the untreated-infected control groups, which was an average of 580.8 [+ or -] 64.57 p,m (Fig. 4D-F). The group treated with PZQ presented a slight reduction in the granuloma diameter, but not a significant one (Fig. 4C and F).
We have found that [beta]-lap interferes in the life cycles of the S. mansoni acting against: skin schistosomula, lung schistosomula, young worms and adult worms. In the therapeutic regimen adopted, [beta]-lap caused significant reductions in loads of worms and hepatic tissue eggs, number and size of granulomas and changed the pattern of oogram. For this evaluation we used the BH strain, which is native from Minas Gerais and predominates in endemic regions in Brazil (Zuim et al., 2012), a country with about six million people infected and another 25 million at permanent risk of contracting schistosomiasis (Lambertucci, 2010).
When a drug is used to suppress egg-laying, the oogram should demonstrate a higher percentage of mature eggs, as a result of previous ovipositions (Pellegrino et al., 1962). This occurred in our study when mice harboring adult worms were treated with [beta]-lap. In this group there was an exchange of percentages between immature and mature eggs, and a significant increase in dead eggs. In the other experimental groups, [beta]-lap did not changed the oogram, since the intervention period occurred during the stages of schistosomula and immature females. In addition, the ovicidal action of PZQ caused inviability of eggs by retraction of miracidia, mostly of mature eggs. The oogram exhibited a greater burden of dead eggs and a lower burden of mature eggs, corroborating with the results of Mantawy et al. (2011).
The [beta]-lap-induced reduction in the burden of eggs and granulomas in hepatic tissue could be attributed to its schistosomicidal activity on female worms at different stages (Table 1). This effect results in a smaller number of granulomatous reactions and liver fibrosis in the course of infection.
Our results indicate that [beta]-lap offers protection against liver injury induced by eggs. The histopathological examination revealed less inflammation of parenchyma and reduction in the size of granulomas when the intervention occurred concomitantly with full maturation of females, when oviposition had already started and eggs had begun to accumulate in liver tissue. At this stage of infection, the granuloma is a delayed hypersensitivity reaction mediated by [TCD.sub.4.sup.+] cells (Pearce and Macdonald, 2002). These cells trigger the Thl response, with macrophage activation and subsequent synthesis of 1L-2,1L-6, TNF-[alpha] and IFN-[gamma], nitric oxide (NO) and citrulline, determining the extent of inflammation (Modolell et al., 1995; Wynn and Cheever, 1995). In the present study, the reduction in the size of the granuloma can be attributed to the anti-inflammatory activity of [beta]-lap, which inhibits the migration of neutrophils and reduces the synthesis of TNF-[alpha], IL-6, NO and NO-sintetase (Liu et al., 1999; Tzeng et al., 2003; Sitonio et al., 2012). This suggests that [beta]-lap favorably modulate the granulomatous immune response and that therapeutic intervention for a longer period could result in an early resolution of the granuloma. In contrast to PZQ that although decreasing the number of eggs and granulomas in the liver tissue, during the acute phase of infection, did not decrease the size of granulomas, these findings corroborating with the results of Aires et al. (2012).
[beta]-Lap is triggering huge interest for its powerful therapeutic properties against various diseases, including cancer, diabetes, obesity, and cardiovascular disease (Dong et al., 2009; Hwang et al., 2009; Kim et al., 2009). Moreover, [beta]-lap has shown, both in vitro and in vivo, to have antimalarial properties against strains that are both sensitive and resistant to chloroquine (Andrade-Neto et al., 2004; Perez-Sacau et al., 2005), and also to be active against Trypanosoma cruzi (Menna-Barreto et al., 2007; Salas et al., 2008; Ferreira et al., 2011), fungi (Medeiros et al., 2010), both gram positive and gram negative bacteria (Loureno et al., 2011; Kenneth et al., 2012; Silva et al., 2009) and to inhibit the replication of the HIV-1 virus (Li et al., 1993). It is suggested that these biological activities of [beta]-lap derive from the inhibition of DNA topoisomerase activity or the impairment of DNA repair through reactive oxygen species (ROS) generation via NAD(P)H: quinine oxidoreductase 1 (Krishnan and Bastow, 2000; Reinicke et al., 2005; Bey et al., 2007; Siew et al., 2012). However, information on the schistosomicidal activity mechanism is still not understood. Remarkably, it is well known that ROS generation can induce cytotoxicity and tissue injuries. The increase in ROS and [H.sub.2][O.sub.2] subsequently depletes the glutathione reserve by oxidizing reduced glutathione (GSH) to oxidized glutathione (GSSG), which could alter the activity of vital enzymes or proteins in S. mansoni, resulting in lipid peroxidation and protein disorganization, thereby affecting the parasites' ability to protect themselves from free radicals, resulting in their death (Azza et al., 2005; Kuntz et al., 2007; Seif el-Din et al., 2011). Therefore, the worms' antioxidant defense mechanisms may constitute a good target for chemotherapy (Azza et al., 2005). The most obvious effect of host ROS on the schistosome is on the parasite surface (Mei et al., 1996). The schistosome tegument contains numerous mitochondria, which could be the main target of [beta]-lap. [beta]-lap affects the mitochondrial functioning by redox cycling-mediated oxidation (Witte et al., 2004), inducing internucleosomal fragmentation of DNA, release of cytochrome c, and activation of caspase-3 (Li et al., 1999a, 1999b; Menna-Barreto et al., 2007; Salas et al., 2008). Recently, Lorsuwannarat et al. (2013) have suggested these mechanisms work as an action route of 5-hydroxy-2-methyl-1,4-naphthoquinone, known as plumbagin, against S. mansoni adult worms. According to Zhai et al. (2002), mature adult worms have a higher anti-oxygen free-radical effect and other biochemical kinds of toxicity than the juvenile worms, leading to a higher mortality rate in juvenile worms than in the mature adult worms of S. mansoni. However, [beta]-lap acted similarly in all the development stages evaluated here.
In contrast to PZQ, which causes an increase in the permeability of the membranes of schistosome cells leading to calcium-producing sustained muscular contraction, spastic paralysis and tegument damage (Doenhoff et al., 2008, 2009), [beta]-lap, in tumor cells and in the ileum, promotes the efflux of this ion, suggesting that the increase in plasma [Ca.sup.2+] is responsible for its apoptotic effect (Tagliarino et al., 2001; Bentle et al., 2006). This antagonistic effect is, therefore, an important target for future [beta]-lap research regarding the calcium channels of S. mansoni.
Naphthoquinones, which include [beta]-lap, have been shown to be active against other phases in the development of S. mansoni. Pinto et al. (1997) evaluated naphthoquinone derivatives that might topically inhibit the penetration of S. mansoni cercariae. From the derivatives studied, 15 naphthoquinones blocked penetration by cercariae when applied to the tails of mice 24 h prior to infection through the tail. In the group exposed to [beta]-lap, blocking was 100%. In the present study administration of [beta]-lap 24 h after exposure to cercariae reduced the worm burden. Studies conclude that topical formulations that block cercarial penetration may be used as prophylactic drugs in endemic regions (Pinto et al., 1997; Kasny et al., 2007; Haas et al., 2008). Furthermore, naphthoquinones derivatives are also active against the intermediate host of S. mansoni. These compounds kill adult snails and the spawn of B. glabrata (Lima et al., 2002a,b; Ribeiro et al., 2009).
In view of the limited aqueous solubility of [[beta]-lap (Lindenberg et al., 2004), formulations have been developed to ensure greater bioavailability and biological activity, the use of lower doses and a convenient administration route, as well as to control minor side effects (Li et al., 2006; Khong et al., 2007; Yang et al., 2008). In view of the moderate activity of [beta]-lap against S. mansoni, the use of new formulations provides the prospect of studies aiming to increase activity and shed light on the action mechanism, and to use this drug as a prototype for the synthesis of new naphthoquinones derivatives with potential schistosomicidal properties.
Received 24 May 2013
Received in revised form 28 June 2013
Accepted 9 August 2013
Conflict of interest
The authors have declared that there is no conflict of interest.
This work received financial support from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Fundafao de Amparo a Pesquisa de Pernambuco (FACEPE) and Universidade Federal de Pernambuco (UFPE).
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Andre de Lima Aires (a,b), Eulalia Camelo Pessoa Azevedo Ximenes (c), Vanessa Xavier Barbosa (c), Alexandre Jose da Silva Goes (c), Valdenia Maria Oliveira Souza (a), Monica Camelo Pessoa de Azevedo Albuquerque (a,b), *
(a) Laboratorio de Imunopatologia Keizo Asami (UKA), Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil
(b) Centro de Ciencias da Saude - Departamento de Medicina Tropical, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil
(c) Centro de Ciencias Bioldgicas - Departamento de Antibioticos, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil
* Corresponding author at: Departamento de Medicina Tropical and Laboratorio de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235, Cidade Universitaria, Recife 50670-901, Pernambuco, Brazil. Tel.: +55 81 2126 8484; fax: +55 81 2126 8485.
E-mail address: firstname.lastname@example.org (M.C.P.d.A. Albuquerque).
Table 1 Effects of [beta]-lapachone and praziquante on worm burdens, eggs and average number of the granulomas in mice harboring different developmental stages of S. mansoni, euthanized 60 days after infection. Administration: Groups Average worm burden days post-infection/ development stage Total Reduction (%) [beta]-Lapachone (50 mg/kg/day for 5 consecutive days) 1[degrees] Treated 15.63 [+ or -] 2.56 * day/skin Control 22.29 [+ or -] 2.29 29.78 schistosomula 14[degrees] Treated 14.57 [+ or -] 1.62 * day/lung Control 23.2 [+ or -] 2.38 37.2 schistosomula 28[degrees] Treated 18.17 [+ or -] 2.32 * day/young worms Control 24.14 [+ or -] 2.54 24.7 45[degrees] Treated 13.0 [+ or -] 2.44 * day/adult worms Control 21.75 [+ or -] 3.95 40.22 Praziquantel (50 mg/kg/day for 5 consecutive days) 45[degrees] Treated 1.22 [+ or -] 0.44 * day/adult worms Control 22.86 [+ or -] 3.08 94.66 Administration: Average worm burden days post-infection/ development stage Female Reduction (%) [beta]-Lapachone (50 mg/kg/day for 5 consecutive days) 1[degrees] 7.37 [+ or -] 1.18 ** day/skin 9.71 [+ or -] 1.11 24.09 schistosomula 14[degrees] 6.85 [+ or -] 0.89 * day/lung 11.0 [+ or -] 1.0 37.7 schistosomula 28[degrees] 8.66 [+ or -] 1.21 * day/young worms 11.14 [+ or -] 1.21 22.26 45[degrees] 6.0 [+ or -] 1.15 * day/adult worms 9.87 [+ or -] 1.45 39.2 Praziquantel (50 mg/kg/day for 5 consecutive days) 45[degrees] 0.57 [+ or -] 0.53 * day/adult worms 9.0 [+ or -] 1.41 93.66 Administration: Number of eggs/g tissue days post-infection/ development stage Hepatic x [10.sup.3] Reduction (%) [beta]-Lapachone (50 mg/kg/day for 5 consecutive days) 1[degrees] 8.29 [+ or -] 1.23 * day/skin 12.32 [+ or -] 2.42 32.71 schistosomula 14[degrees] 7.31 [+ or -] 1.19 * day/lung 12.62 [+ or -] 1.67 42.07 schistosomula 28[degrees] 9.62 [+ or -] 1.27 * day/young worms 14.72 [+ or -] 1.62 34.64 45[degrees] 6.81 [+ or -] 0.86 * day/adult worms 13.1 [+ or -] 2.21 48.01 Praziquantel (50 mg/kg/day for 5 consecutive days) 45[degrees] 4.69 [+ or -] 0.73 * day/adult worms 13.68 [+ or -] 2.11 65'78 Administration: Granuloma schistosomotic hepatic days post-infection/ development stage Average number Reduction (%) [beta]-Lapachone (50 mg/kg/day for 5 consecutive days) 1[degrees] 8.87 [+ or -] 1.88 * day/skin 17.6 [+ or -] 3.05 49.6 schistosomula 14[degrees] 9.85 [+ or -] 2.11 * day/lung 20.40 [+ or -] 2.40 51.7 schistosomula 28[degrees] 12.0 [+ or -] 1.63 * day/young worms 19.8 [+ or -] 1.92 39.39 45[degrees] 8.0 [+ or -] 1.41 * day/adult worms 19.4 [+ or -] 2.4 58.76 Praziquantel (50 mg/kg/day for 5 consecutive days) 45[degrees] 6.14 [+ or -] 1.21 * day/adult worms 19.80 [+ or -] 2.39 68.99 Values are expressed as mean of 10 mice t SD; groups were tested against their respective control using Student's t-test. * p < 0.001. ** p < 0.01.
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|Author:||Aires, Andre de Lima; Ximenes, Eulalia Camelo Pessoa Azevedo; Barbosa, Vanessa Xavier; Goes, Alexand|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Feb 15, 2014|
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