Ecological, Physiological and Biochemical Adaptation in Helminth: Trends in Evolution of Anthelminthic Chemical Agents

This review discusses the landmark anthelminthic substances – traditional medicine components, different categories of pluripotential synthetic and naturally occurring compounds. Much attention is paid to the sections devoted to recent discoveries (Emodepside, Monepantel, Derquantel, Tribendimidine) and to some promising works. The review is also focused on some aspects of molecular mechanisms of action of anthelminthic substances and of helminth adaptation to anthelminthic substances, and on alternative worming treatment options. Based on the systemic analysis of particular features of the chemical structure of anthelminthic substances, the hypothesis on the viability of the targeted search for such compounds among the derivatives of conditionally progenitor cyclic hydrocarbons – benzene, indene, naphthalene, 1Н-cyclopenth [a]-naphthalene , anthracene and phenanthrene – by alternating absolutely unsaturated and saturated structures, including heterocyclic analogues containing nitrogen, oxygen and sulfur and different substitutes and functional groups, was voiced.


Introduction
It is well-known that humans occupy a certain niche in the ecosystem and, standing at the top of the food chain, they interact constantly with environmental pathogenic microand macro-organisms. Human evolution involved fighting different diseases, including helminthoses. Even now helminthoses are still dangerous parasitoses of humans, animals and plants. Chemotherapy and prevention are of high priority in control of these diseases.
The entire variety of anthelminthic drugs available on the market (both single-and multi-multicomponent), with narrow to broad activity ranges, are based on compounds belonging to a limited set of relatively safe and highly efficient active ingredients (substances) [1,2]. Anthelminthic substances are synthetic non-organic and organic or natural compounds. In the latter case, compounds are largely the products of secondary bacterial, fungal and plant metabolism and may be used both in native and in chemically modified form [3]. However, the intensive administration of anthelminthic preparations leads to development of resistance, which is one of the ways of biochemical adaptation of helmiths to the first-and second-order environmental changes (the host and ecological niche occupied by the host, where certain stages of the complex-cycle helminth metamorphoses take place), which necessitates regular renewal of the range of chemical agents, among different measures.
This review is aiming to classify anthelminthic substances by their origin and to outline the prospects of search for new anthelminthic chemical formulas, besides alternative anthelminthic treatment methods.
Numerous folk remedies [7] were created and included into reference books of ancient and medieval philosophers Advances in Pharmacology and Pharmacy 2(2): 30-45, 2014 31 and healers. For instance, the Ancient Egyptian papyrus of Ebers (dated approx. 1550 B.C.) contains information on human snail fever and helminthes [6]. Hippocrates described some worms in his works (dated approx. 500 B.C.) and introduced the terms of helminthos and ascaridos (in Greek, the diseases caused by helminthes and ascarids, respectively) [8]. Ancient Roman physician Galen (approx. 129-200 A.C.), one of the founders of pharmacology, suggested the methods to make infusions and ointments (e.g. with plant extracts) called Galenic drugs; he also introduced the notion of active substances [9]. Avicenna's Canon of Medicine (980-1037 A.C.) mention malaria and many helminths, e.g. Dracunculus [10]. These reference books abound in recommendations as to healing diseases caused by parasite worms, e.g. administration of wormseed sage in ascaridosis.
The anthelminthic effect of the wormseed sage (Artemisia cinae) is due to the natural compound of santonin. It is a sesquiterpenic lactone first extracted in 1830 by German pharmacist Kahler (known as "apotheker Kahler") and pharmaceutical student J.A. Alms from raw materials of Russian origin [11], and introduced into medical practice in 1838 [12]. However, it was not until 1963 that its chemical structure was established [13]. Many other plants (pomegranate, European aspen, male fern, tansy, certain chamomiles, tobacco, pumpkin, garlic) were found to possess anthelminthic properties [9,14,15]. For example, filixan for treatment of cestodiases is derived from the male fern root extract (Dryopteris filixmas) [1]. Structural formulas of some anthelminthic vegetal substances are provided below:

Inception of the Planned Chemotherapy
The idea of using chemical compounds to treat animals and humans was proposed by hermetists (in the 4 th to 16 th centuries), in particular, by iatrochemists (iatrochemistry, or therapeutic chemistry, from the Greek iatrós, meaning physician) who were engaged in drug search and preparation (16 th / 18 th centuries), applying the chemical substances known at that time [16], mostly the derivatives of Hg, As, Sb, Cu, Zn, Fe, S. For instance, Glauber's sault (Na 2 SO 4 10H 2 O) is still used in certain cases as a laxative, to expel intestinal worms [17].
The scientific foundations of chemotherapy of parasitoses (including helminthoses) were laid at the turn of the 20 th century, as a result of establishment of the structural theory of organic and non-organic substances. P. Erlich, Nobel prize winner in 1908, having studied over 600 arsenic compounds, showed the possibility of targeted synthesis of drugs capable of influencing the infinitesimal organisms and proposed the term of 'chemotherapy'. In 1910, as a result of his studies, the arsenic-containing drug of Atoxil [18] against trypanosomiasis (along with salvarsan fighting against syphilis) was put into clinical practice [19]. The beginning of use of suramin, a derivative of urea which can still be prescribed sometimes to patients with onchocercosis and sleeping dropsy (the limitations are due to its high toxicity), goes back to the same period (1916) [1]: 32 Ecological, Physiological and Biochemical Adaptation in Helminth: Trends in Evolution of Anthelminthic Chemical Agents Early in the 20 th century, the simplest chlorine organic compounds, carbon tetrachloride CCl 4 (1921) and hexachlorethane C 2 Cl 6 (1928), started to be used against Fasciola hepatica [1]. Due to their toxicity for mammls, they have been mostly cancelled. However, hexichol (paraxylene chlorine derivative introduced in the USSR in 1964) remains in the list of anthelminthic substances in certain republics of the former USSR and in China (efficient against adult maritas) [20]: Till the mid-20 th century, non-organic compounds of arsenic (As), tin (Sn), stibium (Sb), fluor (F), hydrogen peroxide (H 2 O 2 ), sulfur (S), oxygen О 2 , metallic arsenates -arsenate aluminum and dyad metals (Са, Сu, Zn, Sn), Fe (dyad and triad iron) were widely used as anthelminthic substances, despite the explosive growth in organic substances synthesis. For instance, against mature and immature helminthes, the efficient dose of copper acetoarsenite and calcium arsenate Ca 3 (AsO 4 ) 2 for lambs came to 0.3-0.5 g/capita [21], and the dose of copper sulfate (CuSO 4  5H 2 O) for buffalo calves, 112 mg/kg of live weight [22]. At present, non-organics are not used as anthelminthic substances, due to their high toxicity and emergence of more efficient organic substances, e.g. albendazole, phenbendazole, praziquantel etc., which are used against monieziasis of ruminant and other parasitoses [6]. However, certain organoelemental (e.g. organometallic) compounds (e.g. sodium thioacetarsamid and As 3+ based melarsomin) are used in dogs' heartworm disease (caused by Dirofilaria immitis) [23]. Structural formulas of certain arsenic and stibium-containing substances are provided below [24,25]: Chlorine, copper sulfate, and slack lime are still in the toolbox of veterinarians, cattle breeders and sanitary services for desinfection of reservoirs, stalls and other facilities. Studies are underway to create composite anthelminthic substances, e.g. albendazole and copper pectinate (II) (n = 20-30, molecular weight = 17,000-25,000 Da) [26]:

Benzimidazoles and other heterocyclic anthelminthics
The 1960's saw introduction of efficient pluripotential anthelminthic drugs -benzimidazoles (the most numerous substances), imidazole thiazoles and tetrahydropirimidines into the clinical practice, for treatment of intestinal and non-intestinal nematodoses as well as cestodiases: Thiabendazole (1961), albendazole (since 1972, it has been released for treatment of animal and human echinococcosis) [29], levamisole (1966) [30] and pyrantel (1966) [31] were the first representatives of the named anthelminthic substances. Some of them are still in use (often as part of multi-component anthelminthic substances) [1,2].
Other nitrogen-containing heterocyclic substances emerged in the 1970's, the most important of which were pyrazine isochinolines, e.g. prasiquantel (biltricide, discovered in 1977) administered for treatment of cestodiasis and trematodoses [32]. Prasiquantel showed excellent results and remains in the list of animal and human anti-schistosomiasis drugs [1,2,33]:

Biological Activities of Avermectins
The diversity of avermectins' properties (in addition to anthelminthic, insecticidal and acaricidal activities) arouses interest in searchers [41]. Ivermectin blocks CANCER1-dependent growth in cells of benign and malignant tumors (type II neurofibromatosis, ovarian carcinoma etc.) resulting from inactivation of р21-protein-dependent kinase (CANCER1) [42,43]. Inhibition of the yellow fever virus replication [44] and sporogony in Plasmodium falciparum and Anopheles gambiae by ivermectin has been recently discovered [45]; the anti-TB effect of avermectins was also established [46]. In light of these discoveries, obtaining and study of biological properties of new semi-synthetic derivatives of 16-membered lactones seems to be promising in search for new medicinal substances of different pharmacological groups.

Other Substances
Besides the above basic anthelminthic classes, numerous substances of different chemical origin were obtained: e.g. hygromycin (aminoglycoside antibiotic and anthelminthic agent produced by Streptomyces hygroscopicus) [48], and organophosphoric drugs (metriphonate, citioate, etc.) [1], application of which is limited by low efficiency or significant side effects [6]:

Modes of Action
Availability of a broad range of anthelminthic agents is due to the variety of hosts, heltminths, development of their drugs resistance and some other factors. The processes, in which the anthelminthic substance is involved from its getting into a patient's body to producing the treatment effect, can be consolidated into two groups. The first includes physic-chemical and chemical interaction of the substance with environmental factors (рН, temperature, enzymes etc.) when it moves to the helminth's location in the host's body (Fig. 1), with the respective biochemical and physiological consequences. For instance, 36 Ecological, Physiological and Biochemical Adaptation in Helminth: Trends in Evolution of Anthelminthic Chemical Agents non-fermentative and fermentative changes are possible, as in case of dichlophos that is capable of spontaneous transformation in water solutions into a more active dichlorvos that interacts with the nicotinic receptor (naChR) (14), or nitazoxanide that quickly (in approx. 6 minutes) turns, in the blood plasma under enzyme impact, into active metabolites of tizoxanide and glucoronide tizoxanide that inhibit pyruvate ferredoxine oxidoreductase and thus undermine the helminth's energy metabolism [49]. The second group includes the processes that ensure the anthelminthic agent transportation to target cells via investments (cuticle, tegument) or internal cavities (oral, pharyngeal and intestinal cavities) of the helminth and interaction with these cells (see Fig. 1). By the nature of their interaction with the targeted cell, all anthelminthic agents can be divided into: quick-acting (2-4 hours, they inhibit ionotropic receptors) and slow-acting (1-4 days, they influence metabolic processes) [50] (Fig. 2). Substances included in the first group act as nAChR agonists (imidazol thiazoles, tetrahydropirimidines, pyrazine isochinolines, amino acetonitril derivatives etc.) and antagonists (phenothiazine, spiroindoles), allosteric modulators of GABA А -receptor and glutamate dependent chloride channels (GluCls) (16-membered macrolides -avermectins and milbemycins), GABA-ergic receptor (piperazine) agonists, Са 2+ -channel activators (praziquantel) and Са 2+ -dependent К + -channel SLO-1 activators (emodepside). Slow-acting  -tubulin ligands (benzimidazoles) interfere with its polymerization and the microtubular apparatus formation, which leads to degenerative changes and other metabolic disorders in a nematode's intestinal cells and cuticle. Other drugs of this group are SH-group containing substances (melarsomin); ChEI [carbamates, organophosphoric substances (metriphonate), diphenyl substances (biphenium, nitrozocanate and amoscanate]; cyclo-and lipoxygenases (e.g. DEC that interferes with arachidonic acid metabolism and blocks prostaglandin formation in the host, which leads to capillary constriction and occlusion to microfilaria; it increases microfilaria phagocytosis by vascular walls, lymphocytes and granulocytes; in addition, its effect is obviously related to the inducible NO-syntase); chitinases (in particular, clozantel that also has protone ionophoric activity that is synergic with chitinase inhibition); pyruvate ferredoxin oxidoreductase (nitazoxanide) [50,51].

Efficacy
The helminthosis chemoprevention and chemotherapy cost efficiency largely depends on correctness and appropriateness of methods used combined with efficiency of anthelminthic agents and with allowance for the helminth development cycle, particular features of the host, climate etc. For instance, mebendazole and thiabendazole act on trichinella intestinal forms, but not on incapsulated forms [33,51].
Efficiency comparison for anthelminthic agents of different generations suggests that introduction of new substances was accompanied with the dose reduction and reached the bottom in avermectins [52] (Fig. 3).

Helminth Adaptation and Drug Resistance
Intensive administration of anthelminthic agents is not only accompanied with some adverse biochemical consequences for a patient (negative adverse effects) but also leads to appearance and establishment of the biocide-resistant forms in helminth population. This fact was noted by P. Erlich as early as in the beginning of the 20 th century, and resistance to frequently used anthelminthic substances is now registered universally [52][53][54][55][56][57][58][59][60][61][62][63].
The resistance develops as a result of helminth adaptation to anthelminthic effects. While interacting with the target, it 38 Ecological, Physiological and Biochemical Adaptation in Helminth: Trends in Evolution of Anthelminthic Chemical Agents initiates a cascade of physical, chemical and biochemical events (in addition to those related to the expression of anthelminthic effects) that involve intracellular effectors at different levels (up to the genetic apparatus and its functional activity), and therefore, in addition to the initial target, a modified target may appear and be reproduced (see Fig. 2). Reduction in expression of receptor proteins or its subunits (nAChR agonists), increased P-glycoprotein expression ( [54] and multiple resistance proteins (GluCl allosteric modulators), single nucleotide polymorphism (b-tubuline ligands) play an important part in resistance development [50,61]. In particular, it was experimentally shown in C. elegans nematodes that simultaneous mutation of avr-14, avr-15 and glc-1 genes encoding GluCl -subunits is the reason for significant resistance to ivermectin, due to the reduction in affinity of the substance to the chlorine ion channel. On the contrary, mutation of any two genes of the channel either does not lead to resistance or does lead to an insignificant one [62]. Resistance formation may be accelerated by incorrect anthelminthic agent administration [52][53][54][55] (Table 2).

New Anthelminthic Agents
Resistance onset in pathogenes encourages the search for new anthelminthic agents with a different action mechanism and/or the one typical of the already existing substances (but more efficient) [64][65][66]. For instance, in 2000/2010 emodepside, monepantel, derquantel (veterinary science), as well as tribendimidin and nitazoxanid (medicine) emerged ( Table 2).
Emodepside represents a N-methyl derivative of a 24-membered cyclooctadepsipeptide -the product of Mycelia sterilia fungus fermentation, first extracted from the microbial flora of Japanese camellia leaves by Japanese scientists in 1990 [67]. It is efficient against nematodes in the gastro-intestinal tract and lungs and microfilaria resistant to benzimidazoles, 16-membered macrolides and cholinergic agonists. It is included into newly created drugs recommended for feline and canine nematodosis treatment [68][69][70].
Structural formulas of substances developed in 2000/2010: The efficiency and the action spectrum of the substances administered for anthelminthic treatment depend on many factors: the host (including humans) and the helminth species (their anatomic, histological and physiologo-biochemical properties, e.g. presence of a tegument, body and organ temperature, individual differences); the lifecycle of a bio-or geo-helminth and pathogen localization in the intermediate or definitive host body; the host's position in the ecosystem's food chain, its nutrition and living features (climate, lifestyle etc.) [71,[118][119][120]; bioavailability, physical and chemical properties of the anthelminthic agent and its interaction with the target (receptors, enzymes and other endogenous metabolites and cellular elements) etc., which is (or must be) taken into account in the successful anthelminthic treatment protocol.
Emodepside inhibits muscles function (throat, body and organs involved in egg laying), by increasing conductance of Са 2+ -activated К + -channels (SLO1) in pre-(obviously, to a greater extent) and post-synaptic cells of a nematode's neuromuscular plexus [72] (Fig. 4). However, the sequence of events in this case needs to be specified. They also assume that [73], in addition to the above, the slow-acting signal mechanism (with participation of latrophylin-like pre-synapse and G q -protein receptor) plays a minor part [72,73].
Monepanthel (S-enantiomer, a synthetic product, was discovered in 2008 and admitted to the market in 2010) is a so-called amino-acetonitrile derivative (ААDs) [74]. Its most active metabolite is sulphone monepanthel [75,76], a pluripotential drug against larvae and adult gastro-intestinal helminths that are resistant to the common anthelmintic drugs, is efficient in low doses (2.5-3.5 mg/kg) [77]. The interaction of monepanthel with nemado-specific nAChR Hco-MPTL-1 interfers this receptor function and paralyzes the helminth's muscles [78,79]. Derquantel is a semi-synthetic derivative of paragerquamide А found among natural spyroindoles produced by the mildew Penicillium paraherquei [80,81]. Derquantel inhibits nAChr [82], is a pluripotential anthelmintic agent and is applied in combination with abamectine for treatment and control of different gastro-intestinal nematodoses (including cases of helminths resistance to other drugs), which points to the feasibility of search of anthelmintic agents among such compounds. Tribendimidine is an aminophenyl dimidine derivative of amidantel that was first synthesized in China in 1980 [83] and was introduced into medical practice there in 2004 [84][85][86], it has anti-nematode effect. Tribendimine is included into the L-subtype of nAChR agonists with the same action mechanism as that of levamizole and pyrantel; so it is not prescribed in case of levamizole resistance. However, developers recommend administering tribendimidine instead of benzimidazoles or in combination with them, in case of resistance to the latter [87].
Out of anthelmintic drugs developed during the last decade, it is worth mentioning nitazoxanide [49,88] -a salicylic acid derivative, in which, unlike with salicyl anilides, its residual is bound with the nitrothiazole fragment via an amide bond. As mentioned above, active metabolites of nitazoxanide inhibit pyruvat ferredoxine oxidoreductase (see also [89]). It has protozoacide, anthelmintic and anti-bacterial properties and is used in medicine [2]. By its efficiency, nitazoxanide is inferior to benzimidazoles, but it can be used against benzimidazole-resistant forms [89].
Work on chemical modification of known substances continues. In particular, benzimidazole derivatives, i.e. benzimidazolyl-chalcones (chalcones are flavonoids with an open pyran ring) [90], as well as 5-О-succinoyl avermectin and Gemacs on its basis [91][92][93] have been developed. These so-called horizontal developments are aimed at obtaining analogues with improved physical, chemical and pharmacological properties (e.g. reduction in residual quantities in milk after administration of drugs in the lactation period, etc.).

Some Considerations for Synthetic Search
The search for synthetic anthelminthic substances among the derivatives of conditionally parent hydrocarbons, such as benzene, indene, naphthalene, 1Н-cyclopent[a]-naphtnalene and phenanthrene, by modifying the structure, from absolutely unsaturated to saturated forms, including heterocyclic forms containing N, O, and S atoms, different substituents and functional groups, seems promising. This can be illustrated by classical anthelminthic agents, such as biphenyl, biphenium, and recently discovered substances of monepanthel and tribendimin that contain two and three benzene residuals, respectively. Different saturated and unsaturated cys-hydrindan derivatives (including conditionally heterocyclic analogues, e.g. benzimidazoles) in the form of the CD-fragment of aglycons of cardiac steroids (see section 3.2.1), the divalent residual of cys-1-oxahydrinden-4,5 within 16-membered macrolide anthelminthic agents, the BC-fragment of the tricyclic 1Н-cyclopent [a]-naphthalene (e.g. in the form of santonin containing the condensed 5-membered lactone ring) are hypothetically attractive. In this connection 8, 14-[115], 9,10-and 9,11-secosteroids, e.g. analogues of Torgov seco-diketone, vitamin D 2 [116], 6,7-secoecdysteroids and others may also be promising.

Cardioactive steroids: New Posiblities?
Interestingly, anthelminthic properties were found in certain cardioactive steroids (natural cardenolides and bufadienolides, as well as synthetic 14β-hydroxy-derivatives of estrogens), e.g. asperozide of Streblus asper [110] plant bark and synthetic 14-metoxy-18-methyl-estron [111]. As it is known, positive inotropic effect of cardioactive steroids is associated with inhibition of the membrane Na,K-АТPase [112], but they were also found to produce anti-tumour effect recently [113]. At the same time, modulation of GABA А -receptor activity by neuro-steroids [114] was described. Therefore, the anthelminthic effect of these compounds (through Na,K-ATPase inhibition or otherwise) is yet to be found out.
Anthelminthic effect was found in other synthetic steroid compounds (data will be published in the respective patents).

Alternative Methods for the Helminthosis Control
Biological treatment methods destroying helminth eggs applicable for pastures (nematophagous fungus Duddingtonia flagrans) [94], reservoirs (ascomycete Caryospora callicarpa YMF1.01026) [95], gastro-intestinal tract (nematophagous fungus Pochonia chlamydosporia) [96], immune system reinforcement [97], selection of helminth-resistant breeds and lines should be regarded as alternative approaches to helminthoses control in animals. With discovery of so-called pattern recognition receptors (PRR) that are capable of specific interaction with nematode and flatworm antigens in the inherent and adaptive immune system of mammals, there are prospects to develop vaccines based on resistant helminth tissues [98]. Filariasis treatment using agents acting on Wolbachia pipientis bacteria (cytoplasm symbiont of nematodes) [99], e.g. tetracycline antibiotics such as doxicycline [100] and others, may also be used as a helminthoses prevention and treatment method [101]. It is noteworthy that endo-symbionts were found in more than 90 % of studied nematodes [50,102].

Conclusion
Chemotherapy is central in fighting animal and human helminthoses. Pathogen resistance to known anthelminthic substances necessitates their constant renewal.
A new substance development, be it designing a new compound or identification of properties of any existing compounds, is a time-consuming and expensive process. Application of the diversification (different types of compounds) and the focused (related compounds) screenings, software-based evaluation of anthelminthic activity in silico [117], chemical and genetic trials on model small animals (nematodes Artemia salina, Caenorhabditis elegans, etc.) are intended to accelerate introduction of new substances [50,118]. Further knowledge in molecular action mechanisms of anthelminthic agents and in resistance development would improve planning of helminthoses control protocols.
In our opinion based on codification of known substances by chemical structure, the target search among the derivatives of conditionally parent hydrocarbons -benzene, indene, naphthalene, 1Н-cyclopent[a]-naphthalene, anthracene and phenanthrene by structure variation from unsaturated to saturated forms, including their heterocyclic analogues containing nitrogen, oxygen, sulfur, different substituents and functional groups, may be promising: