Bisphosphonates as Chemotherapeutic Agents Against Trypanosomatid and Apicomplexan Parasites

Introduction

Infections caused by trypanosomatid and apicomplexan protozoa (among them African and American trypanosomes, parasites from the Leishmania genus, and parasites causing malaria, toxoplasmosis and cryptosporidiosis) are among the most widespread human parasitic diseases in the world and are responsible for heavy socio-economic losses, especially in underdeveloped countries.

Therapy against African and American trypanosomiasis, leishmaniasis, malaria, toxoplasmosis and cryptosporidiosis is unsatisfactory. Because of significant toxicity, chemotherapy with some of the drugs available must be carried out under close medical supervision. The development of resistance to anti-protozoal agents and insecticides aimed at the insect vectors of these pathogens and the lack of efficacious vaccines against any of these diseases have made the discovery and development of new drugs a matter of urgency. However, the costs of drug discovery and development in the pharmaceutical industry have escalated during the past 30 years and as a consequence, pharmaceutical companies have focused their research and development efforts on areas where adequate returns are commensurate with such costs. The result has been the complete withdrawal by the major companies from activities directed towards the discovery of drugs aimed at tropical diseases [1]. A possible solution to this problem is the finding of drugs active against parasites but that have been developed for other uses in humans and therefore have been demonstrated to have very low toxicity. A good example of this approach is offered by the bisphosphonates and we will review recent developments in this area. Excellent reviews on other uses of bisphosphonates are available [2-5].

Chemotherapy Against Trypanoso-matid and Apicomplexan Protozoa

American Trypanosomiasis (Chagas’ Disease)

Over 18 million people are infected and over 90 million are at risk of infection by Trypanosoma cruzi, from Mexico in the North to Argentina and Chile in the South of Latin America [6]. Active programs of vector elimination have been in progress for years in some countries such as Argentina, Brazil, Chile and Uruguay [7]. Vector elimination in other countries has been very difficult because of the sylvatic life cycle of the parasite in several regions. It has been estimated that 2 to 3 million individuals have the clinical symptoms that characterize the chronic stage of Chagas' disease, and that 45,000 of them die each year [6]. Even if all the vectors were eliminated the high number of patients already infected makes the search for effective chemotherapy extremely important.

Chemotherapy against Chagas’ disease depends on two drugs, nifurtimox and benznidazole. These drugs are capable of curing at least 50% of recent infections as demonstrated by the disappearance of symptoms and the negativization of parasitemia and serology [8-11]. However, results of treatment trials for acute infections have not been uniform in the different countries [9, 10], probably because of the different drug sensitivity of different T. cruzi strains. In addition, both drugs produce side effects [8, 10, 11]. Treatment duration is another drawback as nifurtimox is given for 30 to 120 days [10-12] and benznidazole for at least 30 days [12].

The usefulness of these drugs for parasitological cure in the indeterminate or chronic stage of the infections has also been questioned; after treatment, serology in most cases remains positive even when parasitemia is absent [10, 12, 13]. Some reports suggest that anti-parasite treatment of chronic Chagasic patients with benznidazole results in fewer electrocardiographic changes and a lower frequency of deterioration in their clinical condition [14]. These findings match well with the fact that benznidazole-treatment of T. cruzi-infected mice induces a late regression of lesions in the myocardium and skeletal muscle [15], and that parasitization of heart tissue is both necessary and sufficient for the induction of tissue damage in T. cruzi infection [16, 17]. These findings stress the need for chemotherapeutic agents that are effective against all strains of T. cruzi, and with fewer or no side effects than those currently available [18].

African Trypanosomiasis

There are two forms of African trypanosomiasis or sleeping sickness: Rhodesian (East African) and Gambian (West African). Rhodesian sleeping sickness is caused by T. brucei rhodesiense and has a very rapid course. Parasites invade the central nervous system (CNS) within weeks and can cause death in several months. The Gambian sleeping sickness caused by T. brucei gambiense has both an acute phase and a chronic phase. The chronic phase has CNS involvement that can lead to death in 2 years or more. At the present time, reports estimate the number of infected individuals at between 300 to 500 thousand [19]. Recent military actions in the region have resulted in that a great percentage of the at-risk population is not covered by diagnostic or vector control measures [20]. There are currently four drugs available for treating African trypanosomiasis in humans: suramin, pentamidine, melarsoprol and eflornithine [21]. Only the arsenical melarsoprol and eflornithine are capable of treating late stage infections although eflornithine is ineffective against T. b. rhodesiense. Administration of these compounds is a problem since they must be administered by intravenous or intramuscular injections [21]. All of them are highly toxic. Drug resistance to arsenical drugs is common while clinical strains refractory to eflornithine and pentamidine have been documented [22]. It is obvious that new and improved drugs are urgently needed.

Leishmaniasis

Leishmaniasis is a group of diseases caused by a variety of Leishmania species. At least 20 species can infect humans originating cutaneous (oriental sore), mucocutaneous (espundia) and visceral leishmaniasis (kala azar) [23]. The latter is the most severe form of the disease, and it is usually fatal if left untreated [24]. This group of diseases affects more than 12 million people with more than 400,000 new cases worldwide per year [25]. In contrast to the other trypanosomatids that affect only developing countries, two species of Leishmania, L. tropica and L. infantum also affect poor health communities in developed countries, such as France, Italy, Portugal and Spain and L. infantum has become endemic in the U.S. [26]. Close to a third of the world population lives in endemic areas and is at risk of contracting the infection [24]. The organic pentavalent antimonials sodium stibogluconate and meglumine antimoniate are the first-line drugs against different types of leishmaniasis. Antimonials are not safe drugs and problems such as the variable composition of the antimony in the drug formulations, and the necessity for a prolonged administration has led to relapses and resistance [23]. Pentamidine and antibiotics like amphotericin B remain as secondary compounds. These drugs are also very toxic. A lower toxicity has resulted from new formulations of lipid-associated amphotericin B [23, 24]. However, the drug is very expensive and its administration requires close medical supervision. Other drugs such as paramomycin, allopurinol and antifungal azoles have been used with variable results [23, 24]. The three best drugs, antimonials, pentamidine and amphotericin B are administered parentally but an oral drug would be most useful for outpatients. The recent success of miltefosine (hexadecylphosphocholine) as a new oral agent for the treatment of visceral leishmaniasis [27] have stimulated interest in this class of compounds previously shown to have antiviral and anticancer activities [28].

Malaria

Four species of malaria parasites cause disease in humans: Plasmodium falciparum, P. vixax, P. malariae and P. ovale. Every year, an estimated 100 million clinical cases of malaria occur worldwide resulting in more than 1 million deaths [29]. Drugs currently available for prevention and treatment of malaria are chloroquine, quinine, pyrimethamine-sulfadoxine, mefloquine, halofantrine, primaquine, dapsone, atovaquone, and artemisinin derivatives [30, 31]. Chloroquine is an effective, inexpensive, safe, and orally available drug and has been the mainstay of malaria chemotherapy for decades. Unfortunately, development of chloroquine resistance is widespread [30]. Resistance is also a problem for quinine, pyrymethanine-sulfadoxine and mefloquine [30]. Mefloquine and halofantrine are also very expensive drugs for many developing countries. Primaquine is the drug of choice to eliminate the pre-erythrocytic stages of P. vivax and P. ovale but is toxic to patients with glucose-6-phosphate dehydrogenase defficiency [30]. Artemisinin derivatives and atovaquone are the most recent additions to the antimalarial drug armamentarium but concerns about their cost, possible toxicity and resistance have limited their use alone. Their combination with other antimalarials is currently proposed [30].

Toxoplasmosis

Human infection with T. gondii is usually asymptomatic. Only the developing fetus and the immunosuppressed patient are at risk of severe disease. With the advent of the AIDS epidemic toxoplasmosis has emerged as a major opportunistic pathogen. Toxoplasmic encephalitis in AIDS patients has been widely reported. Pyrimethamine in combination with either sulfadiazine or clindamycin has been the regimen of choice for treating AIDS- and immunosuppression-related infections. Other drugs recently used for toxoplasmosis are atovaquone, trimetrexate, dapsone, azithromycin and clarithromycin [29]. The efficacy of therapy for toxoplasmic encephalitis in AIDS patients is paliative rather than curative. With the possible exception of atovaquone and azithromycin, no antimicrobial agent has been effective against the tissue cyst form of T. gondii and maintenance therapy becomes necessary [29]. The other big disadvantage of these drugs is that they are very expensive and unaffordable for developing countries.

Cryptosporidiosis

The true incidence of cryptosporidiosis, caused by Cryptosporidum parvum, is unknown, but recent estimates suggest that the organism is a major cause of diarrhea worldwide, causing 250 to 500 million infections annually [32]. In patients with AIDS, infection may result in prolonged life-threatening diarrhea. There is currently no known effective therapy for human cryptosporidiosis [29, 33, 34].

Structure and Mode of Action of Bisphosphonates in Bone

Bisphosphonates are pyrophosphate analogs in which the oxygen bridge between the two phosphorus has been replaced by a carbon with various side chains “Fig. (1)”. Several bisphosphonates are potent inhibitors of bone resorption and are in clinical use for the treatment and prevention of osteoporosis, Paget’s disease, hypercalcemia caused by malignancy, tumor metastases in bone, and other ailments [2-5]. Selective action on bone is based on the binding of the bisphosphonate moiety to the bone mineral [2-5].

Fig. (1). Structure of pyrophosphate and commonly used bisphosphonates.

Some bisphosphonates, such as clodronate can be metabolized to a cytotoxic, nonhydrolyzable analog of ATP by mammalian cells [35], and one inhibits the osteoclast vacuolar H⁺-ATPase [36]. Alendronate has been shown to inhibit protein tyrosine phosphatases [37, 38]. Since these enzymes are involved in many signal transduction pathways, their disruption could interfere with osteoclast activity. However, the more potent nitrogen-containing bisphosphonates or aminobisphosphonates, such as pamidronate, alendronate, ibandronate, and risedronate, are not metabolized [35], and act by a different mechanism that can lead to osteoclast apoptosis [39]. Over the past two or three years, several groups have narrowed the site of action of the potent aminobisphosphonates to the mevalonate pathway [2, 40], or more specifically, to an inhibition of protein prenylation.

The pathway of sterol biosynthesis from mevalonate includes the synthesis by prenyl transferases of isoprenyl-pyrophosphate intermediates, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) [41] “Fig. (2)”. These isoprenyl groups can be transferred to a cysteine residue within carboxy-terminal motifs present in several classes of proteins, including the family of GTP-binding Ras, Rho, Rac and Rab proteins and nuclear lamins [42-45] in a reaction catalyzed by at least three distinct cytoplasmic prenyl protein transferases [45]. Post-translational modification of proteins with C15 farnesyl or C20 geranylgeranyl groups appears to be essential for the localization of these proteins to membranes and hence their biological function [45-47]. Inhibition of protein prenylation by substrate inhibitors of prenyl protein transferases of by inhibitors of mevalonate or isopentenyl pyrophosphate synthesis (such as lovastatin, mevastin, and phenylacetate) has a profound effect on cell morphology [48], cell replication [49, 50], and intracellular signal transduction [51] and can lead to induction of apoptotic cell death [52. 53]. It has been shown that apoptosis induced by bisphosphonates in J774 macrophages is associated with the inhibition of post-translational prenylation of proteins such as Ras and that this effect can be inhibited by the addition of components of the mevalonate pathway such as farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP) [54].

Fig. (2). Overview of the pathway for isoprenoid biosynthesis in trypanosomatid and apicomplexan parasites. The DOXP pathway, present in apicomplexan parasites, is indicated with a light blue background while the leucine incorporation mechanism present in L. mexicana [84] is indicated with a gray background. The mevalonate pathway present in most eukaryotes in indicated with a pink background. The lower part of the scheme shows the synthesis of ergosterol known to occur in trypanosomatids. Inhibitors are indicated in red and enzymes in blue.

Very recently, the mode of action of aminobisphophonates in osteoclasts has been elucidated. Van Beek et al. [55] proposed that they inhibit either dimethylallylpyrophosphate/isopen-tenylpyrophosphate isomerase or FPP synthase. Martin et al. [56] proposed that aminobisphosphonates act as (aza)carbocation pyrophosphate transition state analogs of the native geranylpyrophosphate carbocation and 3 groups independently demonstrated that indeed, aminobisphosphonates do inhibit FPP synthase in osteoclasts [57-59] with IC₅₀s in the sub-micromolar range. In much earlier work, aminobisphosphonates were also shown to be active against a plant FPP (or GGPP-) synthase [60, 61]. Moreover, several crystal structures of bisphosphonate herbicides bound to FPP synthase were obtained by Tarshis [62]. These results all indicate that aminobisphosphonates are strong inhibitors of FPP synthase and thereby of protein prenylation.

Bisphosphonates as Antiprotozoal Agents

The bisphosphonate 1-hydroxynonane 1,1-diphosphonate and other analogs were first shown to inhibit the in vitro growth of Entamoeba histolytica at high concentrations (IC₅₀ = 0.3-1.74 mM) [63]. Further work using commercial bisphosphonates and prolonged incubation times showed better activity with risedronate, CGP 48048, zoledronate, and pamidronate (IC₅₀ = 35, 38, 41, and 46 µM, respectively) [64]. No clear correlation was found between the inhibitory effect of bisphosphonates on ameba growth and their effect on the amebic PPi-dependent phosphofructokinase (PFK), the proposed target of these compounds [64]. Other bisphosphonates identified as potential competitive inhibitors of E. histolytica PFK also showed some activity on ameba growth in vitro [65]. Several bisphosphonates also showed inhibition of T. gondii PFK and replication of the parasites in tissue cultures but again there was not clear correlation between these two effects [66]. This suggested that the target of bisphosphonates in these parasites was not the PFK.

Nitrogen-containing bisphosphonates were shown to be active in vitro and in vivo against T. cruzi, without toxicity to the host cells [67]. Pamidronate and alendronate were active against the intracellular forms of the parasite (amastigotes) with a similar IC₅₀ of 65 µM. No toxicity to the host cells, as assessed by observation of detachment, vacuolation and rounding of the cells was detected except for high concentrations of bisphophonates (> 300 µM). Risedronate was much less effective against amastigotes (IC₅₀ _˜ 300 µM). No correlation was found between growth inhibition and inhibition of the vacuolar pyrophosphatase, a presumed target of bisphosphonates [67]. Pamidronate given intravenously to mice with an acute T. cruzi infection completely arrested the development of parasitemia during treatment. These experiments demonstrated that pamidronate can effectively suppress the proliferation of the parasite in vivo as it does in vitro [67].

Aminobisphosphonates were also effective inhibitors of T. gondii growth in vitro [68]. Pamidronate (IC₅₀ = 42 µM), alendronate (IC₅₀ =37 µM), and risedronate (IC₅₀ = 95 µM) were effective in inhibiting intracellular proliferation of T. gondii but again, no correlation was found between growth inhibition and inhibition of the vacuolar pyrophosphatase of these parasites [68].

More recently a series of bisphosphonates was tested on the proliferation of T. cruzi, T. brucei rhodesiense, L. donovani, T. gondii and P. falciparum in vitro [69]. The results showed that aminobisphosphonates have significant activity against these parasites, with the aromatic species having in some cases nanomolar or low-micromolar IC₅₀ activity values [69]. Risedronate had an IC₅₀ of 220 nM for T. brucei rhodesiense and risedronate had an IC₅₀of 490 nM for T. gondii [69]. Bisphosphonates derived from fatty acids were also synthesized and shown to be potent inhibitors against the intracellular form of T. cruzi exhibiting IC₅₀ values at the low micromolar level [70].

Mode of Action of Aminobisphosphonates in Early Eukaryotes

Aminobisphosphonates were shown to inhibit the growth of amebas of the cellular slime mold Dictyostelium discoideum and it was found that the ranking of the aminobisphosphonate drugs in order of potency as inhibitors of Dictyostelium growth and as inhibitors of bone resorption was the same [71]. Small changes in their structure that caused either large decreases or increases in antiresorptive potency gave rise to similar changes in potency for the inhibition of Dictyostelium growth [71-73]. This led to the proposition that the target of aminobisphosphonates in Dictyostelium amebas must be similar to the target in osteoclasts [71-73]. This was recently confirmed by the demonstration that farnesyl pyrophosphate synthase is, as in osteoclasts [57-59] and plants [60, 61], the intracellular target of aminobisphosphonates in D. discoideum [74].

The reaction catalyzed by the farnesyl pyrophosphate synthase is essential for prenylation of proteins and the occurrence of protein prenylation in trypanosomatid and apicomplexan parasites has been demonstrated [75-82]. The mechanism of isoprenoid synthesis, however, differs in apicomplexan parasites from that in mammalian cells and trypanosomatids in that synthesis of isopentenylpyrophosphate occurs predominantly through a non-mevalonate pathway, as occurs in bacteria and plants [83] “Fig. (2)”. It has also been demonstrated that at least some Leishmania [84] can incorporate the leucine skeleton intact into the isoprenoid pathway for sterol production without breakdown first to acetyl CoA “Fig. (2)”. Over the past several years, hundreds of potent protein farnesyl transferase (PFT) inhibitors have been synthesized with the primary goal of developing anti-cancer drugs and some of these compounds have been shown to inhibit the T. brucei recombinant PFT [82] and to inhibit the growth of T. brucei and T. cruzi [77, 78].

Since sterol biosynthesis should also be affected by farnesyl pyrophosphate synthase inhibition (“Fig. (2)”), the effects of one of the more potent bisphosphonates, risedronate, was investigated on sterol biosynthesis in T. cruzi, together with the effects of ketoconazole, a known 14a-demethylase

Fig. (3). Transmission electron micrographs of a whole unstained T. brucei bloodstream forms (A) and T. gondii tachyzoites (B). Numerous electron-dense vacuoles (acidocalcisomes) of different size are shown in these cells. The contrast of a given structure in these images arise solely from their mass density as the preparations were not stained. Bar = 2 µm.

sterol biosynthesis inhibitor [69]. There was clearly a major decrease in epimastigote proliferation in the presence of 100 mM risedronate or 0.3 mM ketoconazole. At the levels investigated, both drugs had strong effects on proliferation, inducing complete growth arrest after 72 hours [69]. Risedronate alone caused a marked reduction in the level of endogenous sterols, which dropped to less than 1/3 of total sterols. Treatment with ketoconazole led, as expected, to the disappearance of the parasite’s 4,14-desmethyl sterols and a concomitant accumulation of di- and trimethylated sterols, particularly 24-methylene-dihydrolanosterol, which became the most abundant sterol in the cells. When the two drugs were used in combination, complete growth arrest and cell lysis was observed and there was a large reduction in the relative proportion of both 4,14-desmethyl and trimethylated endogenous sterols with respect to exogenous cholesterol. This clearly showed that the blockage of endogenous sterol synthesis induced by risedronate was at a pre-lanosterol level. Since there was also no accumulation of squalene detected, these results suggested that risedronate inhibition must be at the pre-squalene level; this is consistent with the idea that farnesyl pyrophosphate synthase is a principal target of the drug. These results are clearly of interest since they represent the first demonstration of the effects of bisphosphonates on sterol biosynthesis in T. cruzi. Similar effects were seen in preliminary experiments with Leishmania mexicana [69].

It has been postulated [67, 68] that the selective activity of aminobisphosphonates on trypanosomatids and apicomplexan parasites could result from their preferential accumulation in the parasites due to the presence of a calcium- and pyrophosphate-rich organelle named the acidocalcisome [85]. This organelle would be the equivalent of the bone mineral to which bisphosphonates are known to bind with high affinity [2-5]. Acidocalcisomes are acidic compartments rich in calcium, magnesium, sodium, zinc, and short- and long-chain polyphosphates, and are present in trypanosomatids and apicomplexan parasites [86-90] “Fig. (3)”. Interestingly D. discoideum has similar organelles [91] and it is possible that the accumulation of aminobisphosphonates in D. discoideum [72] is through their binding to these organelles, also known to have large amounts of calcium, magnesium and polyphosphates [91].

Inhibition of protein prenylation can lead to induction of apoptotic cell death [52, 53]. Although it is not known whether treatment with aminobispohosphonates leads to apoptosis in early eukaryotes, this phenomenon is known to occur in some of these organisms. Apoptosis has been found to occur in D. discoideum [92] and in several trypanosomatids [93-96], and it has been shown that inhibition of protein prenylation in T. brucei using the statin compactin (an HMGCo-A reductase inhibitor) may lead to apoptosis [97].

Other Effects of Bisphosphonates that Could Enhance their Antiprotozoal Activity

It is interesting to note that macrophages (one of the preferred host cells for T. cruzi, and T. gondii, and the principal host cells for Leishmania spp.), like osteoclasts, appear to be particularly susceptible to bisphosphonates which makes them potentially useful as antiarthritic drugs [2]. In addition, it is particularly encouraging to note that in liver and spleen (tissues susceptible to T. cruzi, T. gondii and Leishmania spp. infection) bisphophonate levels may reach concentrations 140 and 814 times higher than in plasma [98].

A secondary effect of aminobisphosphonates that could enhance their antiprotozoal activity is their effect on the host immune system. Of interest are the recent reports indicating that some aminobisphosphonates are able to stimulate, when administered in single-doses, the immunological response of the host [99-103].

An increase of peripheral blood g/d T cells in patients after their first pamidronate treatment in vivo has been demonstrated [99]. Alendronate, ibandronate, and pamidronate induce a dose-dependent activation and expansion of g/d T cells in primary peripheral blood mononuclear cell cultures of healthy humans at clinically relevant concentrations [100]. Following a single dose of aminobisphosphonates an increase in TNF-alpha and a mild increase in IL-6 was seen with all bisphosphonates in vitro, with the greatest effects seen with the highest concentration of both pamidronate and zoledronate. Significant changes in both TNF-alpha and IL-6 were observed within 3 days of a single dose of pamidronate in patients treated for the first time [101]. It was suggested that g/d T cells play a role as a first line of defense against certain bacterial, parasitic and viral infections [104, 105]. So far, only isopentenyl pyrophosphate (IPP), an isoprenoid pathway intermediate, “Fig. (2)”, isolated from Mycobacterium smegmatis was characterized as a “natural ligand” for the g/d T cell receptor [106]. Bisphosphonates are structurally related to IPP and other isoprenoid pathway intermediates. g/d T cells are believed to be involved in host defense against Leishmania in susceptible mice although not in resistant strains [107]. Therefore, these changes could have significant impact in the treatment of leishmaniasis and other protozoal diseases.

Finally, the poor oral availability of some bisphosphonates could be an advantage in the case of C. parvum since this organism is localized in the intestinal mucosa and can be reached by orally-administered bisphosphonates.

Conclusions

In conclusion, recent results suggest that trypanosomatids and apicomplexan parasites possess an organelle, the acidocalcisome, that as the bone mineral, contains large amounts of PPi/calcium complexed in a semi-crystalline state. These parasites also have a farnesyl pyrophosphate synthase. As osteoclasts and D. discoideum, trypanosomatids and apicomplexan parasites are highly susceptible to bisphosphonates. In addition, bisphosphonates have been shown to accumulate in tissues susceptible to infection by some of these parasites, and to possess immunomodulatory effects and very low toxicity. Millions of people have been treated to date with bisphosphonates and since they are already FDA-approved they constitute an attractive group to develop as chemotherapeutic agents against protozoal diseases.

Acknowledgements

We thank the members of the Laboratory of Molecular Parasitology for useful discussions and Brian N. Bailey for help with the structures. Work in our laboratory was supported by grants from the National Institutes of Health, the American Heart Association, and the UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases. RD was supported by a Burroughs Wellcome New Initiatives in Malaria Research Award and S.N.J.M. is a Burroughs Wellcome New Investigator in Molecular Parasitology.

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