Experimental Parasitology 157 (2015) 12e22
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Full length article
Could miltefosine be used as a therapy for toxoplasmosis? Maha M. Eissa a, Ashraf M.A. Barakat b, Eglal I. Amer a, *, Layla K. Younis c a
Department of Medical Parasitology, Faculty of Medicine, Alexandria University, Alexandria, Egypt Department of Zoonotic Diseases, National Research Centre, Cairo, Egypt c Department of Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
No evidence of miltefosine activity against acute toxoplasmosis. Miltefosine antiparasitic activity against chronic toxoplasmosis was demonstrated. Miltefosine caused significant reduction in brain cyst and amelioration of pathological changes. Miltefosine induced morphological and ultra structural changes in Toxoplasma cysts.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 December 2014 Received in revised form 5 June 2015 Accepted 14 June 2015 Available online 23 June 2015
Toxoplasmosis is a zoonotic protozoal disease affecting more than a billion people worldwide. The shortfalls of the current treatment options necessitate the development of non-toxic and well-tolerated, efficient alternatives especially against the cyst form. The current study was undertaken to investigate, for the first time, the potential potency of miltefosine against Toxoplasma gondii infection in acute and chronic experimental toxoplasmosis. Results showed that there is no evidence of anti-parasitic activity of miltefosine against T. gondii tachyzoites in acute experimental toxoplasmosis. However, anti-parasitic activity of miltefosine against T. gondii cyst stage in chronic experimental toxoplasmosis could not be excluded as demonstrated by significant reduction in brain cyst burden. Moreover, considerable morphological changes in the cysts were revealed by light and electron microscopy study and also by amelioration of pathological changes in the brain. Future studies should focus on enhancement of antitoxoplasma activity of miltefosine against chronic toxoplasmosis using formulation based nanotechnology. To the best of our knowledge, this is the first study highlighting efficacy of miltefosine against chronic toxoplasmosis, thus, increasing the list of diseases that can be targeted by this drug. © 2015 Elsevier Inc. All rights reserved.
Keywords: Toxoplasma gondii Miltefosine RH strain Me49 strain Chronic toxoplasmosis Electron microscopy
1. Introduction Toxoplasmosis is a zoonotic protozoal disease affecting more than a billion people. It has a major significance from the perspectives of public health and veterinary medicine. It is caused by Toxoplasma gondii, an intracellular opportunistic protozoan parasite
* Corresponding author. E-mail address: [email protected] (E.I. Amer). http://dx.doi.org/10.1016/j.exppara.2015.06.005 0014-4894/© 2015 Elsevier Inc. All rights reserved.
that is able to infect any warm-blooded vertebrate cell. The prevalence of toxoplasmosis varies greatly around the world. In the United States, approximately 15% of the population tests show seropositive results for T. gondii (Jones et al., 2007). In immune-competent hosts, T. gondii infection results in mild or nonspecific symptoms (Montoya and Liesenfeld, 2004; Lang et al., 2007). However, in immunecompromised patients, it represents serious problem for public health (Kim and Weiss, 2004). As upon reactivation of a latent infection, it can cause retinochoroiditis or encephalitis (Lang et al.,
M.M. Eissa et al. / Experimental Parasitology 157 (2015) 12e22
2007). Additionally, toxoplasmosis is associated with severe congenital defects when primary infection is acquired during the first trimester of pregnancy (Montoya and Remington, 2008). Currently, the most effective treatment regime for toxoplasmosis is the synergistic combination of the antibiotics sulfadiazine and pyrimethamine. Both are folate inhibitors which block both folic acid biosynthesis and metabolism. Therefore, folinic acid should be supplemented to reduce the risk of bone marrow suppression (Montoya and Liesenfeld, 2004; Ku et al., 2009). Moreover, this regimen is commonly associated with many adverse effects such as haematological toxicity, hypersensitivity (McLeod et al., 2006), intolerance (Montoya and Liesenfeld, 2004), bone marrow suppression and teratogenic effects in the first trimester of pregnancy (Degerli et al., 2003; Schmidt et al., 2006). In certain circumstances, clindamycin, a protein synthesis inhibitor, may also be indicated, but its efficacy is limited, requiring the administration of relatively large amounts of the drug. Atovaquone has also been used with some success, although its efficacy has so far appeared to be less than what is seen with the pyrimethamine combinations (Djurkovi-Djakovi et al., 2002). Barakat and Sylvia 2010 stated that among mice infected with parasite, sulpadiazine and clindamycin at any dose combination protected more animals, demonstrating a significant synergistic effect against T. gondii especially in cases of acute infection. A number of other drugs have been used experimentally, but few have reached the clinic. Laboratory studies have demonstrated that, T. gondii strains show significant resistance to a wide range of inhibitors, including those most frequently used. Moreover, exposure to maintenance therapy as parasites periodically cycle between cyst and free living forms potentially provides almost ideal conditions for the selection of drug resistance (McFadden and Boothroyd, 1999; McFadden et al., 2000; Aspinall et al., 2002). So far, there is no available drug that has the ability to eliminate the parasite. Some drugs can only limit the multiplication of the parasite during the active stage of replication. However, once the parasite encysts in the tissues, the drug loses its effectiveness (Innes, 2010). Therefore, the shortfalls of the current treatment options necessitate the development of non-toxic and welltolerated potent alternatives against cyst form. Miltefosine (MILs) (2-[hexadecoxy-oxido-phosphinoyl] oxyethyl-trimethyl-ammonium), an alkyl phospholipid, is metabolically stable analog of the major eukaryotic cell membrane phospholipid phosphatidylcholine (PC) (Van Blitterswijk and Verheij, 2008). It was initially developed as anticancer agent that exerts its activity against a broad spectrum of established tumor cell lines (Wieder et al., 1999). It is licensed for the topical treatment of breast cancer skin metastases. Then, came the evidence of MILs excellent anti-leishmanial activity both in vitro and in experimental animals. Currently, it is the only effective oral treatment approved for human visceral and cutaneous leishmaniasis (Berman, 2005). Miltefosine is being investigated by researchers seeking treatments for infections which have become resistant to existing drugs. Animal and in-vitro studies proved that it has a wide spectrum of biological properties. It possesses anti-bacterial (Llull et al., 2007), anti-mycotic (Widmer et al., 2006), anti-viral (Cugh et al., 2008) anti-helminthic (Eissa et al., 2011a) and molluscicidal activities (Eissa et al., 2011b). Additionally, it has a wide range of properties against metronidazole-resistant variants of Trichomonas vaginalis (Blaha et al., 2006), Giardia lamblia (Eissa and Amer, 2012), Entamoeba histolytica (Seifert et al., 2001) and several free-living amoebas (Walochnik et al., 2002; Polat et al., 2012). Furthermore, Hexadecyl trimethyl ammonium bromide, a compound structurally similar to miltefosine, was found to exhibit potent in vitro activity against Plasmodium falciparum (Choubey et al., 2007). The pharmacokinetics of miltefosine are mainly characterized
13
by its long residence time in the body, resulting in extensive drug accumulation during treatment and long elimination half-lives. Experimental distribution studies following oral administration demonstrated that the uptake of miltefosine in rats and mice was extensive and in a wide range of tissues (Breiser et al., 1987; Marschner et al., 1992; Sindermann and Engel, 2006) such as; liver, adrenal glands, kidneys, spleen and skin. It remains unknown to what extent miltefosine penetrates the human brain. However, substantial miltefosine concentrations were demonstrated in the brain parenchyma and CSF of patients treated for Balamuthia and Naegleria infections (Schuster et al., 2006). Furthermore, Webster et al. (2012) reported successful treatment of immune-comptent patient with granulomatous amoebic encephalitis caused by Acanthamoeba spp. with oral miltefosine. Therefore, in view of the promising efficacy of miltefosine against a wide range of protozoal infections and its ability to successfully reach the brain to treat encephalitis caused by free living amoebas (Schuster et al., 2006, Webster et al., 2012), the current study was undertaken to investigate the potential potency of miltefosine against T. gondii infection in acute and chronic infection model of experimental toxoplasmosis. 2. Materials and methods 2.1. Parasites and their maintenance 2.1.1. Virulent RH T. gondii strain T. gondii RHHXGPRT( ) virulent strain was maintained in the laboratory of the Medical Parasitology Department, Faculty of Medicine, Alexandria University, Egypt, by continuous intraperitoneal passages into laboratory bred Swiss strain albino mice every three days (Mcleod et al., 1988). For animal infection, tachyzoites were harvested from peritoneal exudates of infected mice on the fourth day of infection, debris and host cells were removed by filtration through a sheet of glass wool fibers. The filtrate was washed three times and diluted with phosphate buffer saline (PBS) pH 7.4. 2500 viable tachyzoites/mouse were injected intraperitoneally for induction of acute infection model (Eissa et al., 2012). 2.1.2. Avirulent Me49 T. gondii strain Avirulent Me49 T. gondii strain was maintained in Zoonotic Department, National Research Centre, Cairo, Egypt by passage through Swiss-albino mice fourth a year. For animal infection, mice were inoculated orally by gavages with brain suspension containing T. gondii cysts (10 cysts/mouse). For preparation of brain suspension, eight weeks after infection, mice were sacrificed, brains were removed, small parts of the cerebrum were fixed in 10% formalin for the histopathological study while the remaining parts were homogenized in a tissue homogenizer (Wheaton USA) with 1 ml saline each. For cyst enumeration, 0.1 ml of the brain suspension were placed on a slide and microscopically counted under high power lens (40). The suspension was then diluted to concentration of 100 cysts/ml. Brain suspensions from chronically infected mice were used for subsequent experimental infections (Djakovic and Milenkovic, 2001). 2.2. Drugs 2.2.1. Miltefosine (Virbac) Milteforan® 2% veterinary oral solution was used. It was kindly supplied by Dr. Paolo Bianciardi, Scientific advisor, Virbac, Italy. It was given orally at the same day of infection in a dose 20 mg/kg for five consecutive days for acute infection model while the treatment started 60 days post infection for fifteen days in the same dose for
14
M.M. Eissa et al. / Experimental Parasitology 157 (2015) 12e22
Fig. 1. The survival rate among different groups studied in established acute toxoplasmosis.
Table 1 The mean density of T. gondii tackyzoites/oil immersion field in impression smears of the liver and spleen of the different studied groups in acute infection model.
Group Group Group Group %r1 %r2 F (p)
I II III IV
Liver
Spleen
0.0 ± 0.0 12.50a ± 1.43 4.20ab ± 0.79 12.33ac ± 1.12 66.4 1.36
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Full length article
Could miltefosine be used as a therapy for toxoplasmosis? Maha M. Eissa a, Ashraf M.A. Barakat b, Eglal I. Amer a, *, Layla K. Younis c a
Department of Medical Parasitology, Faculty of Medicine, Alexandria University, Alexandria, Egypt Department of Zoonotic Diseases, National Research Centre, Cairo, Egypt c Department of Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
No evidence of miltefosine activity against acute toxoplasmosis. Miltefosine antiparasitic activity against chronic toxoplasmosis was demonstrated. Miltefosine caused significant reduction in brain cyst and amelioration of pathological changes. Miltefosine induced morphological and ultra structural changes in Toxoplasma cysts.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 December 2014 Received in revised form 5 June 2015 Accepted 14 June 2015 Available online 23 June 2015
Toxoplasmosis is a zoonotic protozoal disease affecting more than a billion people worldwide. The shortfalls of the current treatment options necessitate the development of non-toxic and well-tolerated, efficient alternatives especially against the cyst form. The current study was undertaken to investigate, for the first time, the potential potency of miltefosine against Toxoplasma gondii infection in acute and chronic experimental toxoplasmosis. Results showed that there is no evidence of anti-parasitic activity of miltefosine against T. gondii tachyzoites in acute experimental toxoplasmosis. However, anti-parasitic activity of miltefosine against T. gondii cyst stage in chronic experimental toxoplasmosis could not be excluded as demonstrated by significant reduction in brain cyst burden. Moreover, considerable morphological changes in the cysts were revealed by light and electron microscopy study and also by amelioration of pathological changes in the brain. Future studies should focus on enhancement of antitoxoplasma activity of miltefosine against chronic toxoplasmosis using formulation based nanotechnology. To the best of our knowledge, this is the first study highlighting efficacy of miltefosine against chronic toxoplasmosis, thus, increasing the list of diseases that can be targeted by this drug. © 2015 Elsevier Inc. All rights reserved.
Keywords: Toxoplasma gondii Miltefosine RH strain Me49 strain Chronic toxoplasmosis Electron microscopy
1. Introduction Toxoplasmosis is a zoonotic protozoal disease affecting more than a billion people. It has a major significance from the perspectives of public health and veterinary medicine. It is caused by Toxoplasma gondii, an intracellular opportunistic protozoan parasite
* Corresponding author. E-mail address: [email protected] (E.I. Amer). http://dx.doi.org/10.1016/j.exppara.2015.06.005 0014-4894/© 2015 Elsevier Inc. All rights reserved.
that is able to infect any warm-blooded vertebrate cell. The prevalence of toxoplasmosis varies greatly around the world. In the United States, approximately 15% of the population tests show seropositive results for T. gondii (Jones et al., 2007). In immune-competent hosts, T. gondii infection results in mild or nonspecific symptoms (Montoya and Liesenfeld, 2004; Lang et al., 2007). However, in immunecompromised patients, it represents serious problem for public health (Kim and Weiss, 2004). As upon reactivation of a latent infection, it can cause retinochoroiditis or encephalitis (Lang et al.,
M.M. Eissa et al. / Experimental Parasitology 157 (2015) 12e22
2007). Additionally, toxoplasmosis is associated with severe congenital defects when primary infection is acquired during the first trimester of pregnancy (Montoya and Remington, 2008). Currently, the most effective treatment regime for toxoplasmosis is the synergistic combination of the antibiotics sulfadiazine and pyrimethamine. Both are folate inhibitors which block both folic acid biosynthesis and metabolism. Therefore, folinic acid should be supplemented to reduce the risk of bone marrow suppression (Montoya and Liesenfeld, 2004; Ku et al., 2009). Moreover, this regimen is commonly associated with many adverse effects such as haematological toxicity, hypersensitivity (McLeod et al., 2006), intolerance (Montoya and Liesenfeld, 2004), bone marrow suppression and teratogenic effects in the first trimester of pregnancy (Degerli et al., 2003; Schmidt et al., 2006). In certain circumstances, clindamycin, a protein synthesis inhibitor, may also be indicated, but its efficacy is limited, requiring the administration of relatively large amounts of the drug. Atovaquone has also been used with some success, although its efficacy has so far appeared to be less than what is seen with the pyrimethamine combinations (Djurkovi-Djakovi et al., 2002). Barakat and Sylvia 2010 stated that among mice infected with parasite, sulpadiazine and clindamycin at any dose combination protected more animals, demonstrating a significant synergistic effect against T. gondii especially in cases of acute infection. A number of other drugs have been used experimentally, but few have reached the clinic. Laboratory studies have demonstrated that, T. gondii strains show significant resistance to a wide range of inhibitors, including those most frequently used. Moreover, exposure to maintenance therapy as parasites periodically cycle between cyst and free living forms potentially provides almost ideal conditions for the selection of drug resistance (McFadden and Boothroyd, 1999; McFadden et al., 2000; Aspinall et al., 2002). So far, there is no available drug that has the ability to eliminate the parasite. Some drugs can only limit the multiplication of the parasite during the active stage of replication. However, once the parasite encysts in the tissues, the drug loses its effectiveness (Innes, 2010). Therefore, the shortfalls of the current treatment options necessitate the development of non-toxic and welltolerated potent alternatives against cyst form. Miltefosine (MILs) (2-[hexadecoxy-oxido-phosphinoyl] oxyethyl-trimethyl-ammonium), an alkyl phospholipid, is metabolically stable analog of the major eukaryotic cell membrane phospholipid phosphatidylcholine (PC) (Van Blitterswijk and Verheij, 2008). It was initially developed as anticancer agent that exerts its activity against a broad spectrum of established tumor cell lines (Wieder et al., 1999). It is licensed for the topical treatment of breast cancer skin metastases. Then, came the evidence of MILs excellent anti-leishmanial activity both in vitro and in experimental animals. Currently, it is the only effective oral treatment approved for human visceral and cutaneous leishmaniasis (Berman, 2005). Miltefosine is being investigated by researchers seeking treatments for infections which have become resistant to existing drugs. Animal and in-vitro studies proved that it has a wide spectrum of biological properties. It possesses anti-bacterial (Llull et al., 2007), anti-mycotic (Widmer et al., 2006), anti-viral (Cugh et al., 2008) anti-helminthic (Eissa et al., 2011a) and molluscicidal activities (Eissa et al., 2011b). Additionally, it has a wide range of properties against metronidazole-resistant variants of Trichomonas vaginalis (Blaha et al., 2006), Giardia lamblia (Eissa and Amer, 2012), Entamoeba histolytica (Seifert et al., 2001) and several free-living amoebas (Walochnik et al., 2002; Polat et al., 2012). Furthermore, Hexadecyl trimethyl ammonium bromide, a compound structurally similar to miltefosine, was found to exhibit potent in vitro activity against Plasmodium falciparum (Choubey et al., 2007). The pharmacokinetics of miltefosine are mainly characterized
13
by its long residence time in the body, resulting in extensive drug accumulation during treatment and long elimination half-lives. Experimental distribution studies following oral administration demonstrated that the uptake of miltefosine in rats and mice was extensive and in a wide range of tissues (Breiser et al., 1987; Marschner et al., 1992; Sindermann and Engel, 2006) such as; liver, adrenal glands, kidneys, spleen and skin. It remains unknown to what extent miltefosine penetrates the human brain. However, substantial miltefosine concentrations were demonstrated in the brain parenchyma and CSF of patients treated for Balamuthia and Naegleria infections (Schuster et al., 2006). Furthermore, Webster et al. (2012) reported successful treatment of immune-comptent patient with granulomatous amoebic encephalitis caused by Acanthamoeba spp. with oral miltefosine. Therefore, in view of the promising efficacy of miltefosine against a wide range of protozoal infections and its ability to successfully reach the brain to treat encephalitis caused by free living amoebas (Schuster et al., 2006, Webster et al., 2012), the current study was undertaken to investigate the potential potency of miltefosine against T. gondii infection in acute and chronic infection model of experimental toxoplasmosis. 2. Materials and methods 2.1. Parasites and their maintenance 2.1.1. Virulent RH T. gondii strain T. gondii RHHXGPRT( ) virulent strain was maintained in the laboratory of the Medical Parasitology Department, Faculty of Medicine, Alexandria University, Egypt, by continuous intraperitoneal passages into laboratory bred Swiss strain albino mice every three days (Mcleod et al., 1988). For animal infection, tachyzoites were harvested from peritoneal exudates of infected mice on the fourth day of infection, debris and host cells were removed by filtration through a sheet of glass wool fibers. The filtrate was washed three times and diluted with phosphate buffer saline (PBS) pH 7.4. 2500 viable tachyzoites/mouse were injected intraperitoneally for induction of acute infection model (Eissa et al., 2012). 2.1.2. Avirulent Me49 T. gondii strain Avirulent Me49 T. gondii strain was maintained in Zoonotic Department, National Research Centre, Cairo, Egypt by passage through Swiss-albino mice fourth a year. For animal infection, mice were inoculated orally by gavages with brain suspension containing T. gondii cysts (10 cysts/mouse). For preparation of brain suspension, eight weeks after infection, mice were sacrificed, brains were removed, small parts of the cerebrum were fixed in 10% formalin for the histopathological study while the remaining parts were homogenized in a tissue homogenizer (Wheaton USA) with 1 ml saline each. For cyst enumeration, 0.1 ml of the brain suspension were placed on a slide and microscopically counted under high power lens (40). The suspension was then diluted to concentration of 100 cysts/ml. Brain suspensions from chronically infected mice were used for subsequent experimental infections (Djakovic and Milenkovic, 2001). 2.2. Drugs 2.2.1. Miltefosine (Virbac) Milteforan® 2% veterinary oral solution was used. It was kindly supplied by Dr. Paolo Bianciardi, Scientific advisor, Virbac, Italy. It was given orally at the same day of infection in a dose 20 mg/kg for five consecutive days for acute infection model while the treatment started 60 days post infection for fifteen days in the same dose for
14
M.M. Eissa et al. / Experimental Parasitology 157 (2015) 12e22
Fig. 1. The survival rate among different groups studied in established acute toxoplasmosis.
Table 1 The mean density of T. gondii tackyzoites/oil immersion field in impression smears of the liver and spleen of the different studied groups in acute infection model.
Group Group Group Group %r1 %r2 F (p)
I II III IV
Liver
Spleen
0.0 ± 0.0 12.50a ± 1.43 4.20ab ± 0.79 12.33ac ± 1.12 66.4 1.36
