Programmed cell death pathways as targets for developing antifilarial drugs: Lessons from the recent findings

Abstract More than half a century has passed since the introduction of the National Filariasis Control Program; however, as of 2023, lymphatic filariasis (LF) still prevails globally, particularly in the tropical and subtropical regions, posing a substantial challenge to the objective of worldwide elimination. LF is affecting human beings and its economically important livestock leading to a crucial contributor to morbidities and disabilities. The current scenario has been blowing up alarms of attention to develop potent therapeutics and strategies having efficiency against the adult stage of filarial nematodes. In this context, the exploration of a suitable drug target that ensures lethality to macro and microfilariae is now our first goal to achieve. Apoptosis has been the potential target across all three stages of filarial nematodes viz. oocytes, microfilariae (mf) and adults resulting in filarial death after receiving the signal from the reactive oxygen species (ROS) and executed through intrinsic and extrinsic pathways. Hence, it is considered a leading target for developing antifilarial drugs. Herein, we have shown the efficacy of several natural and synthetic compounds/nanoformulations in triggering the apoptotic death of filarial parasites with little or no toxicity to the host body system.

the infective larvae are transmitted to the new host. 10These larvae then migrate through the circulatory system, eventually reaching the lymphatic vessels where they undergo further development.Over a period of 6-12 months, they grow into adult males, measuring approximately 40 mm, and viviparous females, reaching sizes of up to 120 mm.During their lifespan of 4-6 years, successful mating occurs, resulting in the production of millions of mf. 11W. bancrofti also referred to as bancroftian filariasis accounts for 90% of the disease burden and is prevalent in tropical regions around the globe.On the other hand, Malayan filariasis is confined to Southeast Asia. 6her forms of filariasis include subcutaneous and serous cavity filariasis.Subcutaneous filariasis is caused by Onchocerca volvulus, which primarily affects the eyes and leads to a condition called Onchocerciasis, commonly known as river blindness. 12On the other hand, Loa loa localizes in the conjunctiva of the eye, causing Loiasis. 13The prevalence of Loiasis is concentrated in the rainforest areas of West and Central Africa, with its transmission being reliant on Chrysops flies. 14In contrast, O. volvulus is transmitted by black flies, Simulium sp. 15 According to the data published by the World Health Organization (WHO) in 1995, onchocerciasis was predominantly found in African countries, as well as certain American and Asian countries. 16,17 affected over 17.7 million individuals, out of which 270,000 were blind and 500,000 had severe visual impairment. 18To encounter this scenario, various elimination programs were launched very soon after the consequences of onchocerciasis in the countries of America and Africa like, the Onchocerciasis Control Programme (OCP), the Onchocercia Elimination Programme in America (OPEA) and the African Programme for Onchocerciasis Control (APOC).Following the implementation of the elimination program, the Global Burden of Disease Study conducted in 2017 estimated that out of 20.9 million individuals infected with filariasis worldwide, approximately 14.6 million suffered from skin diseases, and 1.15 million were affected by blindness. 12,19Thus, a new program namely the Expanded Special Project for Elimination of Neglected Tropical Disease (ESPEN) was launched in the African region in May 2016 through the cumulative efforts of WHO and the Regional Office for Africa (AFRO). 20rous cavity filariasis is caused by Mansonella perstans, Mansonella ozzardi and Mansonella streptocerca and it is predominantly characterized by being asymptomatic. 21The symptoms exhibited are non-specific, including pruritus, urticaria, arthralgia, abdominal pain and fatigue.As a result, this disease has been overlooked and neglected, lacking preventive or control programs for the affected population in endemic areas. 22Infections are prevalent in Sub-Saharan Africa, Central and South America and the Caribbean, among which Africa alone reports approximately 114 million cases of M. perstans infection. 23Mf of M. perstans and M. ozzardi can be found circulating in the peripheral blood, while M. streptocerca is restricted to the skin.The length of these mf ranges from 160 to 240 μm, varying by species. 21,22The absence of evidence regarding morbidity, mortality and clinical trials, coupled with contradictory transmission data, has contributed to a scenario where the government has not taken significant action to address this endemicity.
This study focuses on the current global scenario of LF and highlights the advancements made in developing therapeutics to alleviate the impact.LF stands as one of the oldest and most debilitating neglected tropical diseases (NTDs), posing a significant obstacle to the socio-economic development of affected countries.According to the WHO, in the year 2000, LF infection was found in more than 120 million individuals across 72 countries in the tropical and subtropical regions of Asia, Africa, the Pacific, parts of the Caribbean, and South America. 24,25However, despite efforts, there are still 51.4   million individuals suffering from LF in 44 countries. 26As of 2021, a staggering 882 million individuals from LF-endemic zones remained at risk of LF, emphasizing the continued need for preventive therapy. 27The study estimated approximately 487 million people in India, accounting for 55.27% of the global population at risk, are under the LF threat. 26O has taken the responsibility of managing filariasis cases worldwide and has recommended a preventive treatment strategy called mass drug administration (MDA). 28,29After the implementation of the National Filariasis Control Program in 1962, the members of the World Health Assembly developed a national plan to combat these neglected diseases in 1997.Subsequently, WHO initiated the Global Programme to Eliminate Lymphatic Filariasis (GPELF) with the goal of eliminating LF as a public health problem by the year 2020.
The program established two key objectives.The first objective is to disrupt LF transmission by implementing MDA to the entire population residing in endemic areas.This involves the administration of a single annual dose of diethylcarbamazine (DEC) or a combination of ivermectin (IVM) and albendazole (ALB).The second objective aims to improve the living conditions of the affected individuals by providing essential amenities such as hygiene, skin care and increased access to surgery for men with hydrocele, in order to alleviate their sufferings. 27,29Additionally, controlling measures targeting the vector population were also implemented.In 2000, the Global Alliance to Eliminate Lymphatic Filariasis (GAELF) was established to support GPELF by facilitating advocacy, coordinating partnerships and mobilizing the resources. 30Over the period of 20 years following the national plan, results are not satisfactory.There are very few countries with little success achieved as per the desired level.According to the data published by WHO in 2021, approximately, 9 billion treatments were given to more than 935 million people for a period of 21 years but there are still 44 countries worldwide including India where LF is considered a serious public health problem and the MDA program remained in ongoing status. 27So, an effective and compliant strategy or drug candidate or drug target must be introduced now to overcome the detrimental disease as soon as possible.
Several reports of fatalities 31 and visual loss 32  as cream has become a common tool for diagnosing onchocerciasis, as it causes the appearance of pruritic papules in the death of mf. 35,36However, Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC) have authorized the oral use of DEC, along with IVM, and ALB as anti-filarial drugs.
Currently, these are the only options to treat LF and are widely used despite their significant limitations, increasing resistance and high cost.
Due to the compromised efficacy of the existing therapeutic options caused by side effects and limitations, there is an urgent requirement for the development of a new drug that can effectively target all stages of filarial nematode infections.Reports from Mexico indicate the reappearance of O. volvulus mf due to their resistance to DEC. 37,38 44 ).Additionally, the emergence of resistance to ML in Dirofilaria immitis was first reported by Hampshire in 2005. 45The rapid rise of anthelmintic resistance in the field of veterinary has become a matter of concern for livestock affecting the human populations as well.Resistance to benzimidazoles was found to be associated with Tyr 200 substitution in W. bancrofti when treated in combination with IVM 46 although no history of resistance was found when treated alone. 47ltiple research reports suggest that MDA may contribute to the resurgence of the infection, but it cannot be attributed as the sole cause for this upsurge. 48,49With the current scenario, there is a potential risk that other nematodes may develop resistance to these drugs in the near future, leading to a reemergence of the disease that could affect the population on a larger scale, particularly impacting the health of adults and children in the endemic areas. 50mpared to subcutaneous and serous cavity filariasis, LF is considered as the most debilitating one.Recent epidemiological data highlights that a substantial population (519 million) in South-East Asia remains at a high risk of LF despite the extensive implementation of MDA. 26 The lack of effectiveness of MDA is also evident in India, where 174 LF-endemic districts have been identified. 51,52erefore, it is crucial to prioritize the development of new therapeutic drugs that are resistant-free, affordable and effective, while simultaneously identifying new targets to eradicate both mf and adult parasites, including the assurance of safety of the human host.Exploring the immunological defence of the host and targeting apoptosis would be effective options to fulfil the need.
4][55] Herein, this article enumerates the present status of the efficacy of the antifilarial medications and their potency in exploiting apoptosis as a target for the therapeutic intervention of LF.

| CURRENT S TATUS OF ANTIFIL ARIAL DRUG S AND DRUG TARG E TS FOR TRE ATING LYMPHATIC FIL ARIA S IS
Several drugs have been developed throughout the decades to treat filariasis but their action kinetics are different providing different targets to explore.The following section provides the data about the anti-filarial chemotherapeutics which have been characterized as per their targets.

| Cytoskeleton disruptors
ALB, mebendazole, flubendazole, oxibendazole and fenbendazole all the active derivatives of benzimidazole (product of benzene and imidazole) are well known for anthelmintic action but have a history of slow absorption through gastrointestinal tract and teratogenicity. 56Mebendazole and flubendazole have a strong inhibition role in microtubule formation which acts as an inducer to the loss of cytoplasmic microtubules of the tegumental and intestinal cells of nematodes, followed by reduced glucose uptake [57][58][59][60] has provided evidence that anthelmintic benzimidazole competes with colchicines binding site to make bonding with nematode β-tubulin.

| Ion channel blockers
Glutamate-gated chloride channels (GluCls) are restricted only to invertebrates hence it can be a potential target to work against filariasis. 61IVM is currently in use which is a chemically modified naturally produced avermectin B1 which is a macrocyclic lactone (ML) showing activity against a broad spectrum of parasitic nematodes after oral or parenteral administration.It signals to open GluCl channels in maximum number and induces paralysis of pharyngeal pumping.In Caenorhabditis elegans, the glc-1 gene partly encoding the GluCl α subunit is the major target of IVM. 62,63Concurrently, the presence of avr-14 from the uterine wall of female B. malayi and the embryonic stage of B. malayi mf explain the reason behind the suppression of mf production by female worms after the successful avermectin treatment. 64,65However, a study by Dent et al. 66 sighted that mutation of three genes from C. elegans glc-1, avr-15 and avr-14 simultaneously endue resistance to IVM, whereas no or little resistance when two of them were mutated.
Moxidectin and milbemycin oxime belong to the milbemycin family, which is consanguineous to avermectin and has expanded the area of affectivity of MLS as ion channel blockers. 67Piperazine is a GABA agonist acting on the ligand-gated Cl − channels on the synaptic and extra-synaptic membrane of nematode muscles, leading to hyper-polarization of membrane potential while keeping the Cl − channels in an open state resulting in a malfunctioned sinusoidal movement of nematode which is also called flaccid paralysis. 689][70] Though the study of Colquhoun and Sakmann in 1985 minutely explained levamisole, pyrantel, morantel, and oxantel are organic cations of large size and trying to pass through the nicotinic ion channels from the outside can block the selective filter and produce spastic paralysis on nematode. 71,72Amino-acetonitrile derivatives (AAD) like monepantel block nicotinic acetylcholine receptors, [73][74][75] whereas cyclic depsipeptides are responsible for their target to potassium channels and latrophilin receptors. 73

| Metabolic inhibitors
Lactate produced from carbohydrate metabolism is the major energy source for the filarial parasite in which DEC, antimonials, suramin, benzimidazoles and levamisole targets different enzymes on different steps of carbohydrate metabolism and reduce the metabolism to yield a poor amount of energy.Inhibiting the activity of phosphoenolpyruvate carboxykinase, fumarate reductase and succinate dehydrogenase by DEC alters the rate of carbohydrate metabolism 76 while suramin inactivates lactate dehydrogenase, malate dehydrogenase, 77 and malic enzyme 78 in O. volvulus that ceases tri-carboxylic acid cycle.According to Saz and Dunbar, 79 a very high dose of antimonial stibophen is required to block the activity of phosphofructokinase (PFK) in filariae Litomosoides carinii, Acanthocheilonema vitae and Brugia pahangi by targetting glycolysis.Again DEC, benzimidazoles, and levamisole confirmed their role as glucose metabolic inhibitors by altering glucose uptake, inhibiting glucose transport, and declining glucose utilization by shifting towards homolactate fermentation respectively. 80Interconversion of folate derivatives in nematode is obstructed by the action of DEC and suramin on the enzymes catalysed by the whole folate metabolic process; catastrophic to nematode due to retarded purine synthesis.In particular, suramin, a highly toxic drug, catalysing the inhibition of dihydrofolate reductase (DHFR) in O. volvulus and NADP-dependent 10-formyl FH 4 -dehydrogenase in B. pahangi 80,81 but DEC mediates several inhibitions to different enzymes viz., serine hydroxymethyltransferase, 5, 10 methylene FH 4 reductase, methylene FH 4 dehydrogenase, 5 formyls FH 4 cycloligase, 10 formyls FH 4 dehydrogenase and glutamate formiminotransferase in folate metabolic pathway. 80,821][82][83][84][85] In addition, mevinolin, an inhibitor of HMG-CoA reductase significantly blocks the synthesis of geranyl geraniol, ubiquinone and dolichol in filariid B. pahangi. 80,86

| Arachidonate metabolism blockers
Antifilarial drugs that target arachidonate metabolism have been investigated.DEC has emerged as a notable nematocide due to its ability to suppress the metabolism of arachidonic acid.It achieves this by blocking cyclo-oxygenase (COX), which hinders the production of prostaglandin and prostacyclin-potent inflammatory responses. 68,87DEC also blocks leukotriene synthase, a significant mediator of immune response, converting it into leukotriene. 68,88,89zin et al. 90 have reported that the induction of DEC does not inhibit 5-lipooxygenase.

| Oxidative stress inducers
Antifilarial drug DEC can block arachidonic acid metabolism, whereas aspirin non-steroidal anti-inflammatory drugs inhibit prostaglandin H synthase.According to Singh et al., 91 the combined effect of both drugs increases the level of nitric oxide (NO) and decreases the GSH and peroxidase levels to induce oxidative stress.

| Anti-Wolbachial therapy
The Discovery of Wolbachia endosymbiosis with several filarial nematodes gave new hope to invent novel strategies in the field of anthelmintic therapies. 92Doxycycline has an antibacterial property with the potential to reduce the plasma level of vascular endothelial growth factor-A (VEGF-A) which is a key regulator in hydrocele development of lymphatic filarial patients. 93According to Sanprasert et al., 94 combined therapy of doxycycline and DEC first depletes the Wolbachia and then kills the parasite with the potentiality of DEC.
To avoid the development of resistance to antibiotics for Wolbachia, rifampicin and doxycycline application had been declined to the threshold level 94 but these antibiotics have some limitations in application to children and pregnant mothers. 95Though clinical trials of antibiotics like doxycycline and rifampicin either in single or in combinational use have achieved numerous successful outcomes 96,97 there are also reports of a resurgence of W. bancrofti after 24 months of doxycycline and rifampicin combined therapy. 98Thailand studies have published the data revival of infection of mf and adult bancroftian filarial after 6 months of combined therapy of doxycycline and DEC.  and ALB combined provides microfilaricidal activity. 100Other studies performed using DEC 6 mg/kg along with ALB 400 mg showed a high reduction of adult W. bancrofti. 101,102Subsequent studies carried out in American Samoa starting from 2001 and spanning over a period of 6 years, involving combined MDA of ALB and DEC, demonstrated a significant reduction in the prevalence of W. bancrofti antigenemia.The prevalence dropped from 11.5% in 2001 to 0.95% in 2006. 103ALB alone is worse in comparison to IVM but combined with IVM it has shown a higher mf reduction rate along with a significant clearing ability of mf from night blood. 104,105Makunde et al. 106 demonstrated that IVM + ALB therapy is safe and endurable in treating bancroftian filariasis when coinfected with onchocerciasis.Report from a study of nine villages in South India of re-emergence of W. bancrofti after the successful MDA with an annual single dose of IVM 400 μg/kg with DEC 6 mg/kg rejected the new theory of combinational chemotherapy in treating filariasis but the sustainable result came out when suitable vector control measure all-night landing catch (ANLC) method had accessed against C. quiquefasciatusis. 107Interestingly, several clinical trials on W. bancrofti-infected communities also showed that there were no differences in efficacy between IVM alone and combination therapy with DEC 108 and again in between IVM alone with IVM + ALB treatment. 109

| Life cycle inhibitors
Targeting and blocking the life cycle of the filarial parasites are crucial strategies in reducing transmission and infection burden.
Analysis of numerous epidemiological and clinical trial studies has revealed that the components of MDA have limited efficacy against both mf and adult filarial parasites.Furthermore, the effectiveness of these components varies among different species causing filariasis (Figure 1).1][112][113][114][115] Antibiotics such as doxycycline and rifampicin have demonstrated better results in eliminating human parasites that harbour Wolbachia as an endosymbiont. 94,96,97However, these antibiotics have been found to be ineffective against bovine or rodent parasites.Studies have also reported instances of mf and/or antigenemia reemergence months after treatment, indicating the inability of chemotherapeutic agents to fully block the life cycle of filarial parasites.Failures of drugs like DEC, ALB and IVM are not limited to filarial parasites alone, as similar reports have been observed with other parasite species as well.DEC, for instance, has shown no efficacy against schistosomiasis-causing parasites.[121] F I G U R E 1 Schematic representation of the potential efficacy of different chemotherapeutics across the developmental stages of filarial parasites reside in the human host (Wuchereria bancrofti and Onchocerca volvulus), bovine host (Setaria cervi) and rodent host (Acanthocheilonema viteae).elegans. 122,123Apoptotic cells after the formation of apoptotic bodies are engulfed by macrophages.They never release any substances like lytic enzymes or oxidizing molecules that may induce a local inflammatory response.

| Apoptosis in filarial worms: extrinsic and intrinsic pathways
The filaricidal actions used on the parasites have resulted in the low concentration of Superoxide dismutase (SOD), catalase and Glutathione Peroxidase (GPx) enzymes acting as a vital antioxidant defence mechanism. 124GSH is a sulfur-containing protein that carries reactive electrons from the peroxide that get affected and lowers its concentration, due to filaricidal induction whereas glutathione-S-transferase (GST) maintains a higher concentration of ROS in the mitochondria, endoplasmic reticulum, and peroxisome thus resulting in apoptosis. 125g-laying defective (EGL)-1, cell death abnormal (CED)-3, (CED)-4 and (CED)-9 are four essential proteins with their corresponding role as pro-apoptotic and anti-apoptotic proteins mediating the role in apoptosis in filarial parasites. 53EGL-1 and CED-9 proteins represent the bcl2 family, whereas CED-4 is the nematode homologue of mammalian apoptotic protease-activating factor-1(Apaf-1).
During apoptosis, CED-9 is negatively regulated by EGL-1 and fails to show dominancy over CED-4 and CED-3. 122,126In a normal filarial cell, CED-4 dimers are sequestered with CED-9 on the outer surface of the mitochondria and inhibit apoptosis. 127In time, stimulation after an apoptotic induction increased the level of EGL-1 and made bonding with CED-9 by the BH3 domain that in turn disrupting the CED-4-CED-9 complex. 128,129After dissociation of CED-9, two asymmetric CED-4 dimers oligomerize to make a tetrameric apoptosome and this tetrameric structure then recruits proCED-3 molecules 130 followed by CED-3 a cysteine protease becomes activated and executes apoptosis, which is nematode specific 126,127 (Figure 2).Peixoto et al. 131 weref parasite by among the pioneers who provided molecular evidence of apoptosis in the filarial parasite W. bancrofti.
Further studies conducted by Landmann et al. 126 and Roy et al. 132 demonstrated that the CED pathway operates the induction, progression and execution of apoptosis in the filarids.The signalling molecules of CED pathway are analogous to the pathway operative in the free-living nematode C. elegans. 133,134Existence of the CED pathway was found to be operative in both wolbachial (W.bancrofti and B. malayi) and non-wolbachial (Setaria cervi and S. digitata) filarial parasites 53,126,132,135 that indicated this pathway to be a ubiquitous drug target.
Studies on filaricidal-treated parasites by Mukherjee et al. 53,125,136 revealed a rich level of cysteine protease family protein, called F I G U R E 2 Mechanism of apoptosis in filarial parasites.ROSinduced oxidative stress within the filarial parasites activates a major cell death pathway.EGL-1 the apoptosis inducer disrupts the normal CED-4-CED-9 complex form and makes an association with CED-9 to create a pro-apoptotic moiety.Free CED-4 dimers then oligomerize and make tetrameric apoptosomes with proCED-3.Activated and cleaved CED-3 is the ultimate protein responsible for apoptosis in filaria parasites.
caspase, directing a new path which is suitable for filarial apoptosis by activating other inactive proforms by making a cleavage at a specific sequence next to aspartate.The involvement of caspase protein cas-9, cas-8 and cas-3, cytochrome c and poly (ADP-ribose) polymerase (PARP) in the nematode 125,136 indicates both intrinsic and extrinsic pathways are induced in apoptosis.In conclusion, the nematode-specific CED pathway and the newly arise caspase pathway both have fruitfulness in executing apoptosis in the filarial parasite (Figure 3).

| AP OP TOS IS INDUCER S A S ANTIFIL ARIAL AG ENTS
Natural and synthetic compounds have been considered an alternative over a couple of years since they have shown potential in killing the adult filariid by inducing apoptosis.Apoptotic target killing model has been developed in bovine parasite S. cervi 137,138 and S. digitata 139,140 due to their biochemical, immunological and morphological resemblance to W. bancrofti and brugian parasites.

| Anthelmintic drugs
Apart from the most promising targets, MDA-approved anthelmintic drugs also have some inductive properties to execute apoptosis in filarial parasites.Among the drugs, DEC has a crucial role as a metabolic inhibitor.According to Peixoto et al., Shakya et al. and Singh et al., 91,131,141 DEC when used as a single drug or in combination with aspirin illustrates a decisive role in mediating apoptosis.Microtubule assembly preventer ALB and Wolbachia depletory antibiotics like rifampicin and doxycycline when administered have shown tunnelpositive results along with the presence of fragmented DNA and condensed chromatin proving the involvement of apoptosis in the killing of the parasite. 142,143In animals and in vitro observations of tetracycline on B. malayi provide favourable information, Wolbachia depletion is a key to induce apoptosis to filariid. 126

| Phytochemicals
RT-PCR and ROS measurement study revealed that Hydroxycinnamic acid; a phenolic compound isolated as ferulic acid from Hibiscus mutabilis, has a potent antifilarial effect in ROS generation and has significantly decreased the expression of CED-9 that later, in turn, active CED-3 to progress apoptosis in adult as well as mf in bovine filariid S. cervi. 144The tunnel positive study of Nayek et al. 145 showed the advantageous role of the anti-carcinogen Curcuma longa derived curcuminoid, curcumin to deplete the parasitic GST level and improve the ROS generation that induces apoptosis in micro as well as in macrofilariid.Apart from anti-malarial, anti-leishmanial, anti-trypanosomal and anti-nematicidal properties, ursolic acid is a pentacyclic triterpenoid carboxylic acid from Nyctanthes arbortristis leaves, also having efficient anti-filarial activities in inducing CED mediating apoptosis to mf of S. cervi and W. bancrofti as well as to adult S. cervi. 133Azadirachtin is a tetranortriterpenoid phytocompound extracted from Azadirachta indica, elevates the level of superoxide anion and develops oxidative stress that finally leads to apoptotic death of S.cervi. 146MTT results from the study of Mukherjee et al. 146 showed that Azadirachtin causes a significant reduction in worm viability in both mf and adult parasites.Again, Hoechst and Tunnel staining on oleanolic acid-treated S. digitata provide significant evidence of chromatin condensation and apoptotic body formation among all developmental stages of the parasite. 135

| Plant extract
Methanolic leaf extracts of Aegle marmelos, commonly known as beal, are rich in polyphenol which is sufficient to generate oxidative stress at a high concentration and is considered a pro-oxidative and pro-apoptotic agent to microfilariae parasites. 147Mf of B. malayi treated with a high dose of methanolic root extract of Vitex negundo (nirgundi) loses their motility only about 50% while in combination with H 2 O 2 achievingfull motility. 147According to Sahare et al., 148 the root extract of V. negundo contains saponin which is apoptotic in nature.Butea monosperma is well known for its medicinal value as used to treat various ailments in 'Ayurveda', commonly called the flame of the forest or palasa have antimicrofilariae activity against B. malayi. 149Evidence of lipid peroxidation in the B. monosperma leave extract used to treat microfilaria can induce oxidative stress along with the presence of saponin in the extract has validated the relation between oxidative stress with apoptosis. 149,150Cajanus scarabaeoides commonly known as showy pigeonpea has diverse therapeutic value in treating anaemia, smallpox, dysentery, cholera, gonorrhoea and rinderpest as an ethnomedicine.Among the chloroformic, ethanolic, ethyl acetate and petroleum etheric extraction, ethanolic extracts derived from the stem part of C. scrabaeoides are polyphenol enriched and show the most feasible effect when treated against oocytes, mf and adults S. cervi in activating ROS-dependent CED pathway of apoptosis. 54Neem tree belongs to meliaceae family used in traditional medicine for over two millennia by Siddha and Ayurvedic practitioners as anthelmintic, antifungal, antidiabetic, antibacterial, antiviral, contraceptive and sedative.TUNEL-positive apoptotic nuclei in mf and adult S. cervi suggest ethanolic extraction from leaves of A. indica (EEA) exerts an antifilarial role in mediating apoptosis in S. cervi. 55D. immitis, heartworm of dogs is also an apoptotic target for EEA in which 100 μg ml −1 dose EEA can alter the external morphology of the mf with compressed sheath, breakages and holes across the body. 134Novel antifilarial agent n-butanol extract (NBE) of Diospyros perigrena stem bark proficient in generating in vitro efficacy against mf and adult form of S. cervi by upregulating pro-apoptotic proteins and subsequently downregulating antiapoptotic proteins. 151Analysis of tissue damage and hallmark features of apoptosis in treated S. digitata demonstrate rhizome extract of Curcuma zedoaria is significant to induce proapoptotic proteins thus the extract is potent as micro and macrofilaricidal.and PARP along with filarial-specific CED proteins provide evidence that the trans-stilbene derivatives are efficient in triggering apoptosis in filariids. 125Again, trans-stilbene derivatives along with Resveratrol (RSV) exhibit much more potency than alone RSV in mediating apoptotic death in nematode S. cervi. 125Synthesized compound F I G U R E 3 Apoptosis as a target for developing antifilarial drugs.Apoptosisinducing drugs primarily raise oxidative stress inside the parasite body and subsequently activate intrinsic, extrinsic as well as filarial conserved apoptosis pathways, EGL-1/CED-9/CED-4/CED-3 pathway.

DAS et al.
C-cinnamoyl glycoside also has much potential as an antifilarial agent as well as an apoptotic agent that leads to the permanent death of bovine parasite S. cervi and human-affecting filarial parasite W. bancrofti. 155Butylated hydroxyl anisole is a well-known food additive that is used to treate adult S. cervi, showing an adulticidal effect making it more precious.But compared with macrofilaricidal methyl chalcone (MC), MC takes the lead after the parasite's motility and viability test and analysis of GSH level. 156Derivatives of 2, 4-diaminopyrimidine and 2, 4-diamino-s-triazine are Dihydrofolate reductase inhibitors (DHFR) acting as a potent antifilarial agent with evidence from apoptosis detection ethidium bromide-acridine orange (EB/AO) staining proves these derivatives are also responsible for apoptotic damage in B. malayi mf and adult. 157

| Nanoparticles
Nanotechnology is the new future of drug discovery that uses nanoparticles as a treatment option by delivering the drugs to its targets.
Several filaricidal nanoparticles have already been synthesized in the hope that they could be our future drug in treating filariasis like, chitosan functionalized gold nanoparticles which are compatible biologically with the mammalian host and have low allergenicity and biodegradability with no toxic effect.It can upregulate the production of ROS within the filarial parasite resulting in DNA fragmentation hence it is acceptable as an effective nanotherapeutic inducer against filariasis. 160Chitosan and Terminalia chebula extract-based gold nanoparticles are synthesized by following the green method and has a remarkable anti-filariasis effect with evidence of externalization to the membrane phosphatidylserine on treated filarial parasites after dual staining with 6-carboxyfluorescein diacetate and annexin-VCy3.TUNEL positive result also proves its apoptotic nature as filaricidal. 132Another composition of gold nanoparticles with chitosan and extract of Piper nigrum is very active against S. cervi in inducing oxidative stress, an imbalance redox and further activating pro-apoptotic proteins. 161According to Roy et al. 162 BCH (chitosan stabilized) are more significant effector than BEG (ethylene glycol stabilized) and BST (starch stabilized) silver nanoparticles (AgNP) on apoptotic cell death.When applied in high doses approx.250 μL/ mL to S. cervi, most of the mf and adults lose viability.Silver nanoparticles comprise biomolecule tyrosine and natural polymer starch which are highly active in ROS generation and induce apoptosis in the larval stage of filarial parasites but also in filaria causing vector C. quinquefasiatus. 163Other silver nanoparticles synthesized from funicles extract from Acacia auriculiformis exhibit their antifilarial effect with very little concentrated dose, that is, 50% death at only 3.58 μg/mL concentration compared with raw extract of the plant and also significant in triggering oxidative stress that finally mediates apoptosis of filariids. 164The study conducted by Yadav et al. 165 indicated that the raw plant Andrographis paniculata extract is much weaker than the silver nanoparticle synthesized with the same plant extract in killing parasites by triggering apoptosis as well as confirming the success of nanoformulation in the treatment of filariasis.D-glucose and hydrazine composite silver nanoparticle is also a very potent filaricid in generating oxidative attack at a very low dose. 166Report of propidium iodide staining in the degradation of nuclear DNA supports the apoptotic nature of polyvinyl capped silver nanoparticles when treated against S. cervi. 167According to Zafar et al., 168 ALB-copper (II) oxide nanocomposite is much more effective in establishing oxidative stress in comparison to ALB.This study also supports cost-effective nanoformulation which can be our future therapeutic strategy to combat filariasis.
Herein, Table 1 has been specifically designed to list and provide details on all the effective natural and synthetic compounds that have been found to induce apoptosis in filaria-causing parasites.

| WHY AP OP TOS IS?
All the FDA-and CDC-approved drugs are still used in treating filariasis but the severity of killing has declined remarkably.Several strategies with their ongoing status are in an unsatisfactory state with the success rate gradually decreasing along with the reappearance of infection giving rise to a blurred situation worldwide.In this circumstance, apoptosis could play its role as an appreciable target to combat filariasis.
Cytoskeleton disrupters like ALB block microtubule formation and reduce glucose uptake but the efficiency does not reach a satisfactory level when ALB is used in nano-formulation the resulting antifilarial activity is augmented by the synergistic apoptotic impact.
According to Zafar et al., 168 100 μg/mL ALB-CuO nanocomposite was found to be responsible for 47.3 RM value in adult S. cervi, while 87.1 RM value was visualized after treatment of 100 μg/mL ALB alone under dark condition but when using UV light RM value reduced surprisingly.Approximately, two times more mortality was recorded against mf when treated with ALB-CuO nanoparticles at 100 μg/mL concentration in comparison with ALB. 168Moreover, azoles have some teratogenic properties thus there is always a restriction in use while most of the apoptosis-causing agents are plant extract, so there should be no possibilities of toxic side effects, besides, they show a high efficiency as filaricide.Ethanolic extract from C. scrabaeoides is primarily non-toxic to the host along with most proficient and powerful among plant extracts in targeting apoptosis since 50 μg/mL is sufficient to achieve almost 60% mortality of adult S. cervi as well as it has ovicidal properties. 54Another plant extract EEA is also non-toxic and has an LC 50 value of about 47.12 μg/mL against adult S. cervi while against mf it is 24.72 μg/mL and this report makes EEA a highly efficient killer of filarial parasites. 55,134A B L E 1 Apoptosis as a potential target for developing antifilarial drugs.Ion channel blocker IVM targets GluCl channels with several successful trial reports but compared against apoptosis-targeted agents, it has been found that IVM stands far behind in severity as filaricidal.Report from the MTT assay showed about 90% death of W. bancrofti mf when treated with ursolic acid at a concentration of 10 μg/mL and about 70% death when IVM was applied. 164Again, IVM is only successful as microfilaricidal with a little partial efficiency in adults but most of the apoptotic agents have potentiality against all the developmental stages like RSV and trans-stilbene derivatives when treated with mortality achieved at only 8.867 ± 0.82 μg/mL concentration on adult S. cervi but IVM never matched the level, even at 50 μg/mL concentration. 125Also, from another study, where 100% inhibition was reported of adult S. cervi after carbamo (dithioperoxo) thioate derivatives were applied, IVM can only inhibit approx.48% at 100 μg/mL concentration. 154Levamisole another ion channel blocker has species-specific microfilaricidal efficacy only against W. bancrofti and B. malayi but not in O. volvulus along with several histories of resistance not making a potent filaricide. 169Other ion channel blockers like piperazine, pyrantel, morantel and oxantel all are good in executing their action on their respective areas but are limited to their only targets like cattle, sheep, goats and dogs.In humans, these are hardly used against other nematodes, mainly hookworm, also with several limitations so there is a serious need for an apoptotic agent to eradicate human LF.

Efficacy
DEC is one of the most successful antifilariatic drugs that have versatile properties, that is, from blocking arachidonic metabolism, inhibiting carbohydrate metabolic process resulting in induction of oxidative stress but due to several parasite re-emergence or resistance reports as well as due to adverse side effects, a substitute should be needed to kill parasite with the same intensity that DEC can.In this scenario apoptosis inducer, quinolone-fused cyclic sulfonamide 8 L could be used as a substitute as well as a future antifilariatic drug since 8 L is non-toxic to the host and secondly, it is highly active against all developmental stages of S. cervi.According to Mukherjee et al., 53 LD 50 dose was 281.4 μM for adults, 166.9 μM for mf and to the oocyte, it was 17.3 μM and this intensity of action makes 8 L most proficient among synthetic apoptotic agents.Metabolic inhibitor suramin is also a good antifilarial drug but due to its highly toxic nature, it cannot be used frequently as it shows adverse side effects like aching of muscles and joints where all the newly discovered apoptosis targeted candidates have no harmful activity to the host.
For anti-Wolbachia therapy, several antibiotics have been used for a long time to deplete the Wolbachia, so there should be a possibility of resistance among the antibiotics in the near future.Thus, a replacement is mandated and 8 L could help with its beneficial role in efficiency against Wolbachia infection.
Collectively, the aforementioned studies demonstrate that both natural and synthetic compounds capable of inducing apoptosis are highly efficient in reducing mf and adult stages of LF-causing parasites.To further confirm the superiority of apoptogenic filaricidal agents over non-apoptogenic compounds, a comparative study was conducted.As shown in Table 2, the majority of non-apoptogenic filaricidal compounds exhibit lower efficacy compared to apoptosisinducing compounds.Moreover, the autophagy-inducing compounds, such as Epicatechin-3-O-gallate (ECG) and C-cinnamoyl glycoside, demonstrate significantly higher LC 50 values compared to ursolic acid, trans-stilbene derivatives and carbamo (dithioperoxo) thioate derivatives. 113,130,133,145,158,159Additionally, apoptosisinducing compounds like Tetracycline and 8 L exhibit high efficiency in depleting Wolbachia, 53,114 as indicated in Table 2.
All the studies indicated that exploring the host's immune response will be a great strategy with no possibility of developing resistance in the near future as well as the compounds targeting the same have no toxic effect on the host and have high efficiency in killing both the mf and adult filarial parasites by triggering ROS-induced apoptosis.

| DE TERMINATI ON OF AP OP TOS IS IN FIL ARIAL PAR A S ITE S AND SCREENING OF ANTIFIL ARIAL AG ENTS
Effectivity and apoptosis-inducing properties of all the abovementioned antifilarial agents have been discovered experimentally in the last few years.The application of appropriate methodology for determining the induction of apoptosis has been the key to understanding the efficacy of different natural, synthetic/semi-synthetic compounds and nanoparticles against different pathogenic filarial nematodes.Among the techniques to screening the antifilarial agents, cell viability/MTT assay and Trypan blue dye exclusion methods are the primary choices to the researchers (Table 3).Metabolic activity is one of the crucial hallmarks of the living cell. 178  is a simple method to identify fragmentation of DNA in an agarose gel electropherogram. 180In accordance with DFA, in situ DNA fragmentation can be easily detected by TUNEL assay wherein broken double-stranded DNA can be identified in a histological preparation of filarial parasite having exposure to an antifilarial agent.[191] They can establish either proinflammatory or anti-inflammatory milieu within the host, responsible for overt immunopathology.3][194][195] Antifilarial potential of these future therapeutics could be investigated in the context of apoptosis and this could provide us with very insightful directions.

| CON CLUS ION
In summary, analysis of all the data depicted in the available literature supports that targeting apoptosis is mostly effective than other targets for developing antifilarial drugs.Similarly, most of the available antifilarials trigger apoptotic pathways for inducing death in the lymphatic filarial parasites.Herein, Figure 4 summarizes the importance of apoptosis-inducing natural and synthetic products in targeting the filarial parasites, including the adult parasites, emphasizing their significance over the currently available antifilarial in onchocerciasis patients during oral DEC treatment draw a line of limited application.Intriguingly, the study of Taylor et al. and Hutchinson et al. 33-35 scrutinized that the oral administration of DEC is less severe than the application of DEC lotion throughout the body since it experiences severe pruritus, oedematous papular rash and sometimes vascular eruption along with fever, dizziness, headache and lymphadenitis.Nowadays, this alternative use of DEC | 2821 DAS et al.

2. 6 |
Combinatorial chemotherapy ALB, IVM and DEC are the most popular therapeutic choice since the 20th century and approved by the FDA and CDC.Depending on the complexity of the disease, these drugs are sometimes used solely and sometimes in a group of two or three in different combinations like combining DEC with aspirin and achieve the beneficial role of aspirin in the induction of oxidative stress.While combined groups | 2823 DAS et al. such as DEC + ALB and IVM + ALB gave an idealistic opportunity to aim different targets at a single exposure and also for quick action replay, there was an expectation of no new resistance.Treating LF with co-infection of Wolbachia combinational therapy of doxycycline with DEC also proved the success behind the cooperation of combined drugs and their targets.IDA is a new triple-drug-based treatment therapy as recommended by WHO, by combining IVM, DEC and ALB and in comparison, to the two-drug regimen, the trial of IDA against W. bancrofti in Papua New Guniea showed a low success in sterilizing adult parasite for a period of only 3 years 99 than DEC Experiments based on evidence have proposed the idea of exploring the natural process of innate response to eradicate infective pathogens has given us the idea to target the host natural immune response and promote the defence system to eradicate lymphatic filariasis.DEC has given evidence of inducing apoptosis in vitro and killing the parasite efficiently.Infected cells undergo programmed cell death called 'apoptosis' in which the infected cells or dead cells are removed by the host immune system to maintain the homeostatic balance.During the apoptotic process, the cytoskeleton gets modified resulting in membrane blebbing, chromatin condensation and degradation of DNA into smaller fragments thus resulting in a significant decrease in cell volume.H. Robert Horvitz was the first, who worked on apoptosis and developed a complete pattern of programmed cell death after thorough observation of living cells of C.

4. 4 |
Synthetic compoundsSynthetic cyclic sulfonamide, sultam, sultone and cyclic sulfonates are biologically very active compounds exhibiting antiviral, antifungal and antiparasitic activities.153Among these, quinolonefused cyclic sulfonamide (4,7-dimethyl-3,4,7,8-tetrahydro-3λ6-[1   ,2]thiazino[4,3-f]quinoline-3,3,8-trione) 8 L is a hybrid compound synthesized by fusion of coumarin and quinolone and is imperative to accumulate ROS that increases oxidative stress and later signals for apoptosis by triggering both extrinsic and intrinsic pathways against all the developmental stages of S. cervi.Moreover, 8 L is also beneficial against Wolbachia infection. 53More precisely, the activity of 8 L is more efficacious than IVM along with its non-toxic nature to mammalian hosts that target apoptosis makes it the most promising drug candidate among all filaricidal compound.According to A. Gucchait et al., 154 compound 4a among the series of synthesized carbamo(dithioperoxo)thioate derivatives deliberate as most acceptable in inducing apoptosis to the oocyte, mf and adult stage of S. cervi after the flow cytometry analysis of oocyte and mf, and western blotting of apoptotic protein of adults.The involvement of apoptotic mediator caspase 3, caspase 8, caspase 9, cytochrome c After measuring the TrxR activity in 2,4-dinitrochlorobenzene (CDNB) treated S. cervi mf, it has cleared CDNB targets TrxR, imbalance the cellular redox homeostasis followed by mitochondrial membrane lipid peroxidation and protein carbonyl formation that finally initiates intrinsic pathway of apoptosis. 158According to Mandvikar et al., 159 Im10 (5 -benzylidene-2-imino-3-(4-phenylthiazol-2-yl)thiazolidin-4-one), a thiazolidine derivative has much more potential than DEC to trigger ROS-mediated apoptosis in treated B. malayi mf and adult.

MTT ( 3 -( 4 , 5 -
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow tetrazolium salt and it turns into purple-coloured formazan upon the action of the cellular mitochondrial reductase/NAD (P)Hdependent oxidoreductase enzyme that designates cell viability.162On the other side, live cells do not retain trypan blue dye while dead cell usually accumulates the dye due to loss of membrane permeability and appear as blue.Alteration in the nuclear morphology due to chromatin condensation can be easily monitored using fluorescent nuclear stains such as Hoechest, DAPI, Propidium Iodide, AO/ EtBr etc. Changes in the membrane architecture specifically the position of phosphatidyl serine are a signature of cells going into apoptotic death.179This could be easily diagnosed by a fluorescence stain namely FITC-annexin-V.All these techniques are primarily qualitative but the introduction of flow cytometry has enabled the quantitative detection of the apoptotic parameters (Table4).For example, oocytes or mf undergoing apoptotic changes upon treatment with a drug can be very accurately examined for the quantitative induction of apoptosis and the proportion of cells in early and lateapoptosis stages, necrotic cells and live cells can be acquired.Loss of the integrity of the genetic material is a characteristic feature of programmed cell death.Drug-induced damage in the DNA can be efficiently determined using DNA fragmentation assay (DFA) which TA B L E 2 Comparative efficacy of apoptogenic and non-apoptogenic filaricidal compounds.
therapeutics.In the filarial parasites, programmed cell death is an important phenomenon, typically executed by the CED pathway and the activation of the signalling molecules like CED-9, EGL-1, CED-4 and CED-3 are induced by the exposure of several chemical and/ or phytocompounds.53,54,134Therefore, targeted induction of the CED pathway is considered an effective strategy in developing antifilarial drugs and therefore apoptosis or the apoptotic pathways are considered efficacious targets for developing antifilarials.In this consequence, it can be concluded that targeted activation of apoptosis in filarial parasites through the application of natural antifilarials appears to be the way to achieve the goal of eradicating LF from endemic countries in the near future.AUTH O R CO NTR I B UTIO N S Nabarun Chandra Das: Conceptualization (equal); data curation (equal); formal analysis (equal); investigation (equal); methodology (equal).Pritha Chakraborty: Conceptualization (equal); data curation (equal); resources (equal); validation (equal); writing -original draft (equal).Samapika Nandy: Writing -review and editing (equal).Abhijit Dey: Conceptualization (equal); writing -original draft (supporting).Tabarak Malik: Formal analysis (supporting); supervision (supporting); writing -review and editing (supporting).Suprabhat Mukherjee: Data curation (equal); methodology (equal); supervision (lead).

F I G U R E 4
Apoptosis as an efficacious target in developing antifilarials in comparison to conventional drug targets.
Studies conducted by Awadzi et al., Nana-Djeunga et al. and Osei-Atweneboana et al. 39-43 in the endemic communities in Ghana and Cameroon provided evidence of IVM-resistant O. volvulus.Several studies have explored the prevalence of resistance to benzimidazole, levamisole, IVM and moxidectin in Haemonchus contortus, Trichostrongylus colubriformis and Cooperia spp., which are nematodes affecting sheep, goat and cattle respectively (as reviewed in Howell et al.

Name of antifilarial agent Chemical class Efficacy Targeted filarial worm Postulated mode of action
Screening techniques confirming the efficacy of the antifilarial agents.Techniques determining the apoptosis in filarial parasites.Hoechst 33342 or 2′-[4-ethoxyphenyl]-5-[4-methyl-1piperazinyl]-2,5′-bi-1H-benzimidazole trihydrochloride trihydrate is preferentially bound to the adenine-thymine (A-T) regions of the DNA of live or fixed cells and fluoresces at 460 nm when excited by ultraviolet (UV) light End Labelling technique, where the TdT enzyme labels 3′-hydroxyl terminus of DNA breaks with biotin tagged d UTP.Subsequent mixing of streptavidin-HRP and chromogenic substrate DAB with the labelled DNA breaks generates a brown colour that identifies apoptosis Acridine orange (AO) is a nuclear stain, permeable for both living and dead cells, whereas ethidium bromide (EtBr) stains only dead cells' nuclei.Upon intercalation with DNA, AO in normal cells emit green fluorescence and EtBr in dead cells emit orange fluorescence.Flow cytometry Flow cytofluorimetric analyses utilize Annexin V/Propidium Iodide (PI) double staining technique and summarize the result in a two-dimensional dot plot.Annexin V emits a signal for detecting apoptosis while PI for necrosis and late apoptosis Plots show double negative for Annexin V and PI denotes normal cells and necrosis for double-positive.While positive for Annexin V and negative for PI signifies early apoptosis, negative for Annexin V and positive for PI imply late apoptosis TA B L E 3