Keywords

1 Introduction

Protozoans are microscopic, nonfilamentous protists widespread in most aquatic/soil habitats worldwide, being now considered as a paraphyletic group, with a complex evolutionary history [1, 2]. Most protozoans are heterotrophic organisms that acquire their nutrients from the environment, but there are examples of mixotrophic protozoans, such as Paramecium spp., which can also perform photosynthesis for their metabolic needs [1, 2]. Although such organisms are fascinating due to their multitude of phyla, genera, and species, many of which possess ecological and industrial importance, we will discuss here only the parasitic protozoans that infect humans and animals. They produce diseases which range from mild to moderate, such as those induced by Toxoplasma gondii or Entamoeba histolytica, to more serious conditions (e.g., infections due to Cryptosporidium parvum, Giardia lamblia, Trichomonas vaginalis, Babesia spp.) or very serious and widespread ones, such as malaria (infection due to at least five different species of Plasmodium), leishmaniasis (infectious protozoans are various species of Leishmania), Chagas disease (produced by Trypanosoma cruzi), and African sleeping disease (infection due to Trypanosoma brucei) [3,4,5,6,7,8,9,10,11], etc. Although rare, there are also several fatal protozoal diseases, such as those induced by amoebae belonging to the following three genera/species: Naegleria fowleri [12] Acanthamoeba spp. [13], or Balamuthia mandrillaris [13], for which few effective therapeutic approaches are available so far. Although the 12 protozoans genera which produce human disease are now well studied, there are still few available drugs for effectively treating these conditions. Furthermore, the drugs that are used have been available for decades, with high toxicity and low therapeutic indexes, and more concerning, extensive resistance to these treatment options has developed [3,4,5, 14,15,16,17,18]. Thus, what’s going wrong? Why don’t we have effective drugs for diseases that affect hundreds of millions of people worldwide, considering, for example, that for malaria alone there is an estimate of 229 million infections in 2019 linked to 409,000 fatalities [19]? We will attempt to answer these questions here.

2 Antiprotozoal Drugs in Clinical Use

One of the complications encountered when studying protozoans and approaches to inhibit their growth is related to the fact that most of them have quite complicated life cycles, with many different stages and also more than one host, with the vertebrate (human) being generally just one component in this intricate cycle [4,5,6, 16,17,18, 20, 21]. Taking as example again for malaria, which is provoked by at least five Plasmodium species which infect humans (i.e., P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi), the parasite is transmitted by mosquito bites (usually provoked by Anopheles mosquitoes) to vertebrates, infecting them, in a two-stage process. The first, pre-erythrocytic stage includes the following phases: (1) the plasmodial sporozoites are inoculated from mosquito salivary glands into the host through the bite; (2) the sporozoites reach the hepatocytes in the host’s liver; (3) in the hepatocytes the parasite continues its development leading to schizonts which release into the bloodstream the merozoites, the final pathogenic form of the pre-erythrocytic stage. The second, asexual reproduction cycle (also known as blood-stage infection) includes: (1) the asexual multiplication of merozoites in red blood cells; (2) formation of immature ring stage trophozoites which consume the entire content of the invaded erythrocyte; (3) mature schizonts are formed in this way, which burst the red blood cell and release new merozoites into the bloodstream, which continue and enhance the infection of many other red blood cells. As it can easily be seen from this simplified description, Plasmodia have at least six different stages/phases during their life cycle, with the various forms of the pathogen present in different organs and tissues, but also with many different genes which are expressed in the different phases, and with a substantial capability to evade the host immune defences [4,5,6, 16,17,18, 20, 21]. This situation is also generally complicated for other pathogenic protozoans, which have complex life cycles and different secondary hosts (which can be different species of insects, for T. cruzi, T. brucei, and Leishmania spp.), or even other mammals, for example, Toxoplasma gondii (usually Felidae) [4, 5, 10]. In the case of Cryptosporidium spp., Giardia lamblia, Entamoeba spp., or Trichomonas vaginalis infections it seems that there is not an intermediate host, although these protozoans also have rather complex life cycles [6,7,8,9]. Overall, the complicated life cycles make it challenging to clearly identify promising druggable targets.

A second factor which is associated with difficulties in finding new/effective drugs for these pathogens is related to the fact that some of these organisms are difficult/impossible to grow in culture (e.g., Cryptosporidium spp.) [22] or their diverse stages/forms respond differently to drugs. Furthermore, some of these parasite stages are not at all prone to be grown in culture in order to allow a detailed study of the effectiveness of a drug during various stages of their life cycle [22,23,24,25,26].

Last but not least, many of the protozoan diseases are considered tropical diseases which affect a relatively low number of patients from poor countries. This is a very distorted reality, since as already mentioned, only malaria provokes a huge number of infections and many casualties each year. Furthermore, owing to climate change, the enhance of temperatures in parts of Europe, North America, and Australia may soon create conditions for some of these “tropical” diseases to also appear (or reappear in some cases) in these parts of the world.

Table 1 shows the diseases provoked by protozoans in humans (and farm animals, in the case of Babesia) and the currently used drugs for their treatment. Only the two drugs shown in italics characters in Table 1, Fexinidazole and Nitazoxanide have been released for clinical use in the last 3 years [27, 28]. All other drugs shown in Table 1 were in fact discovered in the ‘30–‘70s (except artemisinin and its derivatives, discovered in the ‘80s) and are characterized by rather high toxicity, low therapeutic index, and many side effects, although they are effective, especially in early phases of infection [4, 14,15,16,17,18]. Furthermore, just a limited number of chemotypes are present in the armamentarium of the antiprotozoal drugs, with the nitroazoles being predominant, followed by the dihydrofolate reductase and dihydropteroate synthase inhibitors [29] (Fig. 1). Although the recent approval of the two new agents Fexinidazole and Nitazoxanide is remarkable and demonstrates that relevant achievements can be obtained, both belong to the same class of nitroazoles.

Table 1 Diseases provoked by pathogenic protozoans and the therapeutic agents used for their treatment
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Fig. 1
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Chemical structure of drugs currently used in the therapy of human diseases caused by protozoans

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3 New Drugs and Compounds in Clinical Trials

Acoziborole (SCYX-7158) (Fig. 2) is one of the few compounds in Phase III clinical trials for the treatment of T. brucei infection, being a benzoxaborole derivative, i.e., a totally new chemotype in the armamentarium of antiprotozoal drugs [30, 31]. Benzoxaboroles possess a range of pharmacological activities [32] in addition to antiprotozoal activity, including anti-bacterial, anti-fungal, antiviral as well as carbonic anhydrase inhibitory action [33,34,35].

Fig. 2
figure 2

Chemical structure of Acoziborole

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Several new generation azoles such as ravuconazole and its prodrug (fos-ravuconazole) seem to be promising anti-T. cruzi agents [36], but there is limited information regarding their clinical trials. It is rather disheartening to see that even for malaria, the worst of the documented protozoan diseases, most of the clinical trials that are registered in EU [37] deal with various vaccine candidates or combination therapies of existing drugs, but do not consider novel chemical entities.

4 Challenges for the Future after the COVID-19 Pandemic

The SARS-CoV-2 pandemic that emerged in late 2019 to early 2020 in China and spread all over the world [38, 39] should teach us that neglected diseases are a Sword of Damocles for the entire planet. The unprecedented (at least since 1918) crisis created by the outbreak of this viral disease demonstrated how susceptible the world is and how unprepared we were to tackle such a situation. As shown in this chapter, the number of protozoans is huge and many of them are poorly investigated and understood. Furthermore, such diseases are not restricted to tropical countries as some of the deadliest can be encountered in milder climates, including various Amoeba species that can provoke meningoencephalitis, which is difficult or impossible to treat with currently available drugs [12, 13]. The number of available drugs is limited, restricted to a low number of clinical classes, and only a few stages of the parasite life cycle, which is by itself rather complex. The antiprotozoal drug targets are also quite limited, and although a relevant number of important discoveries have emerged by use of various ‘omics methods over the previous two decades, there were essentially no significant translational studies from the lab to the clinic. Specifically, with the exception of two nitroazoles (see above), which were approved in the last 5 years, and the benzoxaborole derivative acoziborole (Fig. 2), no new drugs have emerged to treat protozoan-based diseases. What is going wrong? In addition to the challenges outlined above, there is also the perception that these are tropical diseases that will not affect people in affluent countries in which pharmaceutical research and large companies tend to be highly active. However, the tragic events of the last 18 months should remind us that this is no longer the case. Why do drug companies not invest in developing new antiprotozoal drugs, considering that the available ones are of low effectiveness and can have high toxicity? This situation should change, given the many interesting discoveries from academic researchers based all over the world that have emerged over recent decades, many of which are presented in the chapters of this book. By presenting an update of the state of the art in such diseases for nearly all protozoan infections, the current and broad gap in knowledge that needs to be bridged to develop excellent drugs for the treatment and management of these pathologies will become clearer.