Effects of Natural Products on Neuromuscular Junction

Abstract

Neuromuscular junction (NMJ) disorders result from damage, malfunction or absence of one or more key proteins involved in neuromuscular transmission, comprising a wide range of disorders. The most common pathology is antibody-mediated or downregulation of ion channels or receptors, resulting in Lambert-Eaton myasthenic syndrome, myasthenia gravis, and acquired neuromyotonia (Isaac’s syndrome), and rarely congenital myasthenic syndromes caused by mutations in NMJ proteins. A wide range of symptomatic treatments, immunomodulating therapies, or immunosuppressive drugs have been used to treat NMJ diseases. Future research must be directed at a better understanding of the pathogenesis of these diseases, and developing novel disease-specific treatments. Numerous secondary metabolites, especially alkaloids isolated from plants, have been used to treat NMJ diseases in traditional and clinical practices. An ethnopharmacological approach has provided leads for identifying new treatments for NMJ diseases. In this review, we performed a literature survey in Pubmed, Science Direct, and Google Scholar to gather information on drug discovery from plant sources for NMJ disease treatments. To date, most research has focused on the effects of herbal remedies on cholinesterase inhibitory and antioxidant activities. This review provides leads for identifying potential new drugs from plant sources for the treatment of NMJ diseases.

1. INTRODUCTION

The neuromuscular junction (NMJ) is the excitatory chemical synapse between the myelinated motor nerves and skeletal muscle fibers, which utilizes acetylcholine (ACh) as the neurotransmitter. It transforms the nerve action potentials to muscle contraction. This process is fast, lasts milliseconds. Vesicles present in the motor nerve terminals release ACh in the presynaptic membrane. Next, the ACh diffuses across the NMJ and binds to acetylcholine receptors (AChR) on the postsynaptic membrane. Occupation of AChR leads to a transient increase in the permeability of the membrane to sodium and potassium ions. The local end-plate depolarization resulting from this process is known as a miniature end-plate potential (MEPP). Depolarization of the presynaptic membrane leads to an influx of calcium ions into the motor terminal, producing exocytosis of ACh vesicles. The postsynaptic depolarization is known as end-plate potential (EPP), producing action potential of the muscle membrane.

The stimulation of the membrane leads to a cascade of events generating muscle contraction. This transmission ends with the reuptake of acetylcholine from the synapse and its rapid hydrolysis by acetylcholinesterase (AChE) [1].

The NMJ is well developed by 20 weeks of gestation, with the refinement of the postsynaptic membrane continuing through the full term. Muscle-specific receptor tyrosine kinase (MuSK) and rapsyn are responsible for the clustering of acetylcholine receptors (AChR) in utero which are located on the postsynaptic membrane, producing a transient increase in the permeability of the membrane to sodium and potassium ions [2]. The neuromuscular junction, along with its components are shown in Fig. (1).

Fig. (1).

Neuromuscular junction and its components. Acetylcholine is produced from choline and acetyl-CoA by the enzyme choline acetyltransferase in the presynaptic terminal. It activates voltage-gated Ca2+ channels when an action potential reaches the end-plate, allowing Ca2+ ions to pass further into the axon terminal along with the acetylcholine release into the synaptic cleft. The released acetylcholine binds to postsynaptic corresponding receptor α subunit and induces the Na + potential within the myofibers. In turn, it activates the Na+ channel voltage gate receptor through the generation of an action potential. This whole process leads to the activation of further receptors, including dihydropyridine and ryanodine type-1 receptors which in turn release calcium ions from the sarcoplasm reticulum into the cytoplasm. Choline is released after cleavage of acetylcholine by acetylcholinesterase and taken up by prestnaptic terminals via choline transporters. Choline is recycled in the formation of acetylcholine.

Neuromuscular junction diseases are uncommon neurological disorders, resulting from the failure of signal transmission at the neuromuscular or myoneural junction [3, 4]. NMJ diseases are disorders of neuromuscular transmission, including specific syndromes such as myasthenia gravis (MG), Lambert-Eaton myasthenic syndrome (LEMS), congenital myasthenic syndromes (CMSs), as well as botulism. MG is the most prevalent disease with clinical features of skeletal muscle weakness [5]. NMJ diseases clinically present as skeletal muscle weakness in the eyes, face, neck, arms, and legs resulting in drooping of the eyelids (ptosis), double vision (diplopia), difficulties with speech (dysarthria), chewing, swallowing (dysphagia), breathing, walking and fatigue. The main etiology of NMJ disorders is associated with autoimmune, paraneoplastic, hereditary, infectious, and toxic effects, while age and gender distribution vary depending on the diagnosis [6].

Given there is a lack of standard monotype, a typical disease mechanism in a range of NMJ disorders treatment protocols, include diverse therapeutical approaches. Neuromuscular blocking agents, acetylcholinesterase (AChE) inhibitors, and immunomodulatory agents are highlighted as common potential therapies for various NMJ diseases. Natural products such as galantamine, tubocurarine, and derivatives, physostigmine, and several other natural metabolites have shown efficacy in the treatment. In this article, the role of natural products in the treatment of NMJ diseases will be reviewed along with the epidemiology and pathophysiology of NMJ diseases, along with common treatment strategies.

2. EPIDEMIOLOGY OF NEUROMUSCULAR JUNCTION (NMJ) DISEASES

Neuromuscular junction diseases are rare disorders. The prevalence of MG varies from 5.35 to 35 per 100.000 population worldwide, with a mean prevalence of 10 per 100.000 people. MG has been shown to occur up to twice as often in women, especially in the younger age group, which is typical for autoimmune disorders [7]. The estimates are relatively lower in Chile (8.36/100,000), Republic of Ireland (15.12/100,000) and are higher in Korea (19.24/100,000) [8-10]. The prevalence has been trending higher in recent years, whilst remaining stable over time, likely reflecting diagnosis and better treatments leading to a longer lifespan of affected patients [11]. LEMS, however, is a rarer disease, with a mean prevalence of 0.3 per 100.000 people within a range from 0.23 to 0.40 per 100.000. LEMS commonly occurs later in life with male predominance and it is associated up to 50 percent with tumor as a paraneoplastic syndrome [12].

Several NMJ diseases are encountered during infancy. Infantile botulism typically occurs between 3-6 months of age in previously healthy babies and toddlers in the setting of a dirty puncture wound or consumption of improperly canned foods [13]. Transient neonatal myasthenia gravis is a relatively short-lived disorder that typically occurs at birth in 15-20% of infants whose mothers have myasthenia gravis, or received magnesium sulfate treatment for preeclampsia [14]. Congenital myasthenic syndromes are rarer and associated with a primary deficit at the NMJ site varying between the presynaptic NMJ, the synapse, and the postsynaptic NMJ such as end-plate deficiency, and they clinically present with recurrent episodic apnea [15].

3. PATHOPHYSIOLOGY OF NMJ DISEASES

Normal physiological neuromuscular transmission is a process that enables the central nervous system to control skeletal muscle movements. The nerve impulses reaching at the presynaptic terminal of the motor neuron cause depolarization, leading voltage-gated Ca2+ channels to open, and ensuing calcium influx into the nerve terminal. This process induces a cascade that begins with phosphorylation of specific proteins called synapsins, which keep vesicles containing acetylcholine (ACh) in a presynaptic actin network which then allows presynaptic membrane fusion and the release of ACh into the junction between the nerve and the muscle cells. Next, these molecules bind specific nicotinic ligand-gated cation channels (ACh receptors or AChRs) on the surface of the receptive muscle fibre, leading to the opening of these channels. In turn, the muscle depolarizes and the opening of voltage-gated sodium channels initiates an action potential and contraction of the muscle cell. Following ACh reduction, channels on the postsynaptic membrane change their conformation and close, consequently repolarizing the cell and causing muscle relaxation. ACh is rapidly degraded by an enzyme called acetylcholinesterase (AChE) to prevent excitation of the muscle repeatedly [19]. The principal components in NMJ are; a nerve terminal that contains ACh, a synaptic cleft containing AChE, and a postsynaptic motor end-plate that contains nicotinic AChRs [20].

The pathogenesis of NMJ diseases is due to impairment in any of the steps described above [16]. In MG, muscle weakness results primarily from the ensuing effects of destructive autoimmune antibodies directed against AchR, muscle-specific kinase (MUSK), lipoprotein-related protein 4 (LRP4), or again in the postsynaptic membrane at the neuromuscular junction. MG is associated with a tumor originating from the thymus gland, thymoma, which occurs in 10–15% of MG, and approximately 35% of patients with thymoma are reported to have MG [17]. Autoimmune MG per se is not a solitary hereditary disease, but is encountered more frequently in other family members due to its autoimmune nature. Ocular myasthenia is a subtype of MG, clinically presenting with the weakness that begins in and remains limited to the muscles of ocular movement and eyelids and approximately two-thirds of MG patients have an entirely ocular presentation at disease onset [18].

Lambert-Eaton myasthenic syndrome (LEMS) is also an autoimmune disease characterized by antibodies directed against the voltage-gated calcium channels (VGCCs) on the presynaptic motor nerve terminal, specifically a subset of the P/Q-type Ca2+ channels involved with neurotransmitter release [21]. The underlying cause of LEMS is small cell lung cancer whichis slightly more than 50% of all cases in the context of paraneoplastic background. In LEMS, the amount of ACh released by the nerve is diminished; however, presynaptic stores of ACh and the postsynaptic response to ACh remain intact. Therefore, voluntary activation or rapid repetitive stimulation increases the release of the amount of ACh, which then permits the generation of the action potential [22]. Congenital myasthenic syndromes (CMSs) are genetic diseases arising from molecular defects most frequently in the muscle nicotinic AchR, but the mutations can also affect presynaptic proteins or proteins functioning in glycosylation [23].

Botulism is an acute acquired neuroparalytic disease caused by botulinum toxin, one of the most poisonous biological substances which is a neurotoxin produced by the bacterium Clostridium botulinum. Botulinum toxin blocks the release of ACh from motor end-plates into the synaptic cleft in cholinergic nerve fibres, consequently inhibiting neurotransmission. The toxin interferes with the vesicle docking and fusion by irreversibly binding to several vesicular and neuronal membrane proteins resulting in inactivation of the process [24]. Botulism has provided considerable insight into the studies regarding the evolution and mode of action of other NMJ diseases and comprises a significant role in medical history. Six clinical forms of botulism have been recognized [25]: classical foodborne botulism, wound botulism, intestinal botulism such as infant botulism, inhalational botulism, botulism of unknown source, and iatrogenic botulism (inappropriate administration of botulinum toxin during its use as a pharmaceutical agent). Time of disease onset, duration of action, symptoms, and other factors in the clinical course varies depending on the serotype of the toxin and the type of botulism, with serotype A responsible for a more dangerous and long-lasting disease [26]. Botulinum toxin can be used to treat several disorders, such as muscle spasticity, blepharospasm, and strabismus [27]. The cosmetic version of botulinum toxin is a commonly used injectable preparation that temporarily reduces or eliminates facial wrinkles with commercial forms marketed under the brand name Botox® [28].

4. TREATMENT MODALITIES IN NMJ

Various drugs, such as succinylcholine and other neuromuscular blocking agents, affect the NMJ [29]. However, treatment options for NMJ diseases are mostly limited to acetylcholinesterase (AChE) inhibitors and immunosuppressive therapies. Acetylcholinesterase (AChE) inhibitors indirectly increase the amount of ACh at the synaptic cleft and strengthen the muscle by preventing the breakdown of ACh. Reduction in ACh hydrolysis by inhibiting the degrading enzyme AChE is the most commonly utilized and effective symptomatic treatment in MG [30]. Physostigmine, pyridostigmine, neostigmine, and ambenonium chloride function as AChE inhibitors, and they are frequently coupled with atropine, a drug that reduces muscarinic side effects of AChE inhibitors [31]. In cases of severe MG which are not clinically resolved by conventional treatments, immunosuppressive drugs are also used. Corticosteroids, azathioprine, mycophenolate mofetil, rituximab, ciclosporin, methotrexate, and tacrolimus are highly efficient immunosuppressive agents [32]. For acute cases, intravenous immunoglobulin

(IVIG) and plasma exchange have also been used. Thymectomy is sometimes undertaken as an oncological procedure when thymoma is detected [5]. Unfortunately, AChE inhibitors do not commonly resolve LEMS, also requiring removal of the lung tumor [21]. The mainstay of management of botulism is supportive care with careful monitorization of respiratory failure, cardiac arrest, and enteric integrity. Passive immunization with a vaccine prior to toxin exposure provides the synthesis of antibodies that neutralize botulinum toxin in the circulatory system. Following exposure to the toxin, rapid administration of antitoxin within hours after toxin exposure can strikingly reduce morbidity, and mortality due to botulism [33]. Supportive treatment in NMJ diseases includes regular physical activity, bodyweight control, avoiding drugs that interfere negatively with neuromuscular transmission, such as D-penicillamine, telithromycin, fluoroquinolones, aminoglycosides, and macrolides [34]. Various neuromuscular disorders and their associated mechanisms are schematised in Fig. (2).

Fig. (2).

Various neuromuscular disorders and associated mechanisms..

5. PROMINENT ROLE OF ALKALOIDS IN NEUROMUSCULAR JUNCTION DISEASES

Alkaloids are an important group of chemical constituents in plants commonly used [35-42]. Today’s special interest in these chemicals relates to their transformation using microorganisms and their enzymes. They are used as natural or de novo compounds. Alkaloids have been used in clinical practice and have been associated with biological activity. Alkaloids play a critical role as a significant factor in biometabolic processes. The regulation of biological metabolism is based on enzyme activities working in concert in sequential pathways [43]. Alkaloids prompting AChE activity are lycorine, galantamine, serine, lobeline, tubocurarine, and nicotine. Given their AChE activities, these alkaloids are preferred for the treatment of NMJ diseases.

5.1. Physostigmine

A pyroloindole alkaloid physostigmine [1,2,3,3a,8,8 ahexahydro-1,3a,8-trimethyl-N-methylcarbamate, (3aS,8aR)-pyrrolo(2,3-b) indol-5-ol] was first isolated from the seeds of Physostigma venenosum Balf. Physostigmine (eserine) increases acetylcholine concentrations, causing stimulation of both muscarinic and nicotinic receptors. Carbamate group in its structure is reasoned to be responsible for cholinesterase inhibition [44].

Parasympathomimetic alkaloid physostigmine, a partial antagonist of curare, was used in a 56-year-old MG patient. Hypodermic injections of physostigmine salicylate (Fig. 3), leading to some minor improvements [45].

Fig. (3).

Chemical structure of physostigmine salicylate.

Cholinesterase activity of physostigmine in blood and tissues has been previously reported [46-52]. The binding constant (KI) and bimolecular rate constant (K'2) were determined in the pons and medulla oblongata, which represent respiratory centers. 50 nM concentrated physostigmine decreased AChE activity in the whole brain, cerebellum, pons, frontal cortex, basal ganglia, and medulla oblongata by 5.7%, 4.6%, 10.1%, 8.0%, 8.4%, and 25%, respectively. The rate of inhibition of AChE in the different sections of the brain is thought to reflect the gray and white matter content [53].

Nicotinic receptors were stimulated with 1 to 10 nM concentrated physostigmine in hippocampal neurons and rat striatal synaptosomes with 0.3 to 300 nM concentrated physostigmine preventing the stimulating effect of nicotine on dopamine release [54, 55]. These activities corroborate the partial agonist effects of physostigmine on nicotinic receptors.

However, due to the short half-life of physostigmine (30 minutes) and undesirable side effects, its application as an anticholinesterase inhibitor for the medical treatment of neurological disorders [48] has been limited. Accordingly, new derivatives and analogues of physostigmine, such as prostigmine (neostigmine) and pyridostigmine (Fig. 4), have been synthesized. In NMJ disorders such as myasthenia gravis and non-autoimmune congenital myasthenic syndromes, neostigmine methyl sulfate and pyridostigmine bromide have been widely used over the last five decades. Their solubility in water depends on the quaternary structure and they have limited ability to cross the brain-blood barrier. Given their rapid metabolism, their clinical efficacy is limited, lasting between 30 to 120 minutes. Carbamate analogues of physostigmine; phenserine, tolserine, cymserine, and phenethylcymserine have been shown to exert longer periods of cholinesterase inhibition [56-58].

Fig. (4).

Chemical structures of neostigmine and pyridostigmine.

Yu et al. conducted a study to evaluate the anticholinesterase activities of phenyl carbamate derivatives of neostigmine methylsulfate (2) and pyridostigmine bromide (4) combined with their corresponding precursors (1) and (3), as well as methyl quaternary derivatives of (−)-phenserine (6), (−)-tolserine (7), (−)-cymserine (8) and (−)-phenethylcymserine (9) (Fig. 5). Phenylcarbamoyl side chain in the analogues 2 and 4 caused significant loss in anticholinesterase activity. Phenylcarbamates compounds 5, 6, 7, 8, and 9 exhibited a preserved or increased AChE inhibitory effect. In addition, compound 16 was administered at a concentration of 10 mg/kg (i.p) to the anesthetized rats. Plasma cholinesterase inhibition occurred rapidly and reached 66% in 60 minutes, gradually dropping to 33% in 24 hours. In addition, no cholinergically mediated central or peripheral side effects were seen [59].

Fig. (5).

Chemical structures of synthesized compounds.

Pyridostigmine has also been used for the treatment of Lambert-Eaton myasthenic syndrome (LEMS) [60-62]. A 52-year-old female patient diagnosed with Lambert-Eaton myasthenic syndrome and treated with intravenous immunoglobulin, pyridostigmine, 3,4-diaminopridine, and azathioprine, with the patient's clinical picture improving upon treatment [63]. In contrast, no traces of clinical course were observed in a 30-year-old female LEMS patient, in response to her treatment with pyridostigmine [64].

5.2. Huperzine A

Huperzine-A (1-amino-13-ethylidene-11-methyl-6-azatri- cyclo [7.3.1.0^2,7] trideca-2(7),3,10-trien-5-one), a sesquiterpene alkaloid first isolated from Huperzia serrata (Thunb.) Trevis. Clubmoss. It is a potent, selective, and well-tolerated reversible inhibitor of AchE. Huperzine A is more selective in the inhibition of AChE than BuChE and also more selective for the G4 form of AchE [65,66].

Mice, rats, and rabbits were used to evaluate the cholinergic agonist activities of Huperzine A and analog Huperzine B (Fig. 6). Neostigmine, physostigmine, and galantamine were used as positive controls to compare the activity. Mouse salivation and anesthetized rabbit EEG pattern test also anesthetized rat sciatic-tibialis, rat isolated phrenic nerve diaphragm, and rat isolated, and chronically denervated hemidiaphragm preparations carried out. The relative magnitude order of therapeutic indices of the studied compounds Huperzine B (26.5) > Huperzine A (23.1) > Neostigmine (8.6) > Physostigmine (3.8) in mice and Huperzine B (294.8) > Huperzine A (72.9) > Galantamine (36.0)> Neostigmine (34.0) > Physostigmine (7.2) in rats. In the light of the results obtained by the researchers, Huperzine A and Huperzine B, which are cholinergic hypofunction activity, have been reported to be effective in the treatment of several peripheral or central nervous system diseases [67].

Fig. (6).

Chemical structures of Huperzine A and Huperzine B.

Tang et al. investigated the efficacy of huperzine A on AChE activity, acetylcholine (ACh) levels, and release, and cholinergic receptors in the rat brain. Rats were treated with 0.1-2 mg/kg i.m. or i.p. The research indicated that huperzine A provided long-term inhibition of AChE for about 360 minutes and ACh levels caused a 40% increase in 60 minutes. After huperzine A administrati on, ACh levels increased at different rates in various brain regions: frontal (125%) and parietal (105%) cortex and slight increments (22-65%) in other brain regions [68].

In a study with 128 myasthenia gravis patients, huperzine A was used in the treatment instead of prostigmine. It was stated in the study that the duration action of huperzin A is longer than prostigmine. In addition, fewer side effects were observed in patients compared to prostigmine [69].

(±)-Huprine Y and (±)-huprine Z (Fig. 7) hybrids of tacrine–huperzine A were evaluated against tacrine and huperzine A for the AChE and BChE inhibition activity. It was stated that (±)-Huprine Y and (±)-huprine Z were more active in human AChE inhibition and showed mixed type AChE inhibitors. Both of the two compounds revealed more selectivity for AChE than BChE. Both compounds were also evaluated ex vivo to address their inhibitory activity of brain AChE. (±)-Huprine Y was found to be more strong than (±)-huprine Z with IC 50 1.09 (0.39–2.98) µmol/kg [70].

Fig. (7).

Chemical structures of Tacrine, (±)-Huprine Y and (±)-Huprine Z.

The synthetic racemic mixture of (+/-)-huperzine A and natural (-)-huperzine A examined for their acetylcholinesterase inhibitory activity both in vitro and in vivo. In in vitro assays racemic (+/-)-huperzine-A (IC50 3x10^-7) was found to have less activity than (-)-huperzine A (IC50 1x10^-7). In vivo assay rats were treated with intraperitoneally 0.1-2.0 mg/kg (+/-)-huperzine A and natural (-)-huperzine A. Both racemic and natural huperzin A showed strong acetylcholinesterase inhibitory activity and long-term effects compared with physostigmine. In addition, any of the huperzine A variants had no effect on choline acetyltransferase activity in the cortex or the hippocampus between 0. 1- 1.0 mg/kg [71].

More recently, Galdeano et al. (2018) evaluated the tacrine and Huprine derivatives' effects on myasthenia gravis. Most synthesized compounds exhibited stronger effects on both enzymes than pyridostigmine. Huprine and triazole analogs (compounds 1-3) (Fig. 8) showed the strongest and selective human AChE inhibitory activity, with IC50 of 59.2 nM, 200 nM, and 7.18 nM, respectively [72].

Fig. (8).

Chemical structures of synthesized compounds.

5.3. Ephedrine

Phenylalanine derived ephedrine, (1R,2S)-2-(methyla- mino)-1-phenylpropan-1-ol (Fig. 9), is the main alkaloid of Ephedra L. species. It has been used for the treatment of myasthenia gravis, as early as 1930 [73].

Fig. (9).

Chemical structure of ephedrine.

Canine intercostal muscle end-plates were analyzed by microelectrode techniques to evaluate the efficacy of ephedrine on neuromuscular transmission. Concentrations < 10^-4 M ephedrine had no influence on neuromuscular transmission, but the quantal content of the end-plate potential augmented by 21%. Analysis of acetylcholine-induced end-plate current noise was used to evaluate the kinetic features of the acetylcholine receptor channel. According to the results ephedrine (10 ^-4 M), decreased the channel conductance by 43%, and at 5× 10 ^-4 M concentration ephedrine enhanced the open time by 23 percent [74].

Mutations in DOK7 underlying recessive congenital myasthenic syndrome (CMS) have been shown. In 15 patients with DOK7 mutations pyridostigmine, 3,4 DAP, and ephedrine combinations were evaluated. Pyridostigmine has been shown to worsen the weakness, 3,4-DAP has a variable response, and ephedrine constitutes a positive response [75].

Cereda et al. evaluated the efficacy of ephedrine in a 60-year-old female patient with idiopathic LEMS. The patient, who used pyridostigmine for 2 years in the past, then 3,4 DAP and IvIg infusion, was then administered ephedrine sulfate at the dose of 200 mg/d. The study concluded that ephedrine may exhibit a benign effect on muscle function at a remote level as a central effect. A relative mismatch between the beneficial clinical advantage and low compound muscle action potential at rest during treatment with single ephedrine was observed in the patient. Consequently, ephedrine with DAP has been noted to have synergistic and beneficial effects [76].

In the study conducted to test whether ephedrine has a direct influence on the NMJ, it was reported that ephedrine does not activate but rather lowers the sub-maximal response to close-arterial injections of acetylcholine. The results showed that ephedrine has an independent action at the neuromuscular junction [77]. Similarly, another study reported that ephedrine increased NMJ transmission by inducing β2-adrenergic receptors and stabilized the structure of the neuromuscular junction [78].

Standard treatment of muscle MG patient worsened, but with salbutamol treatment, increased endurance and respiratory support need to be decreased. Combination of salbutamol with ephedrine showed significant symptomatic improvement in the patient [79].

Wrinten et al. conducted randomized, controlled, double-blind n-of-1 trials to evaluate the potency of add-on treatment with ephedrine on autoimmune myasthenia gravis (MG) patients. Treatment included 25 mg ephedrine twice daily along with pyridostigmine, low-dose prednisone, and azathioprine treatment. Ephedrine has been reported to have positive effects on four MG patients [80]. In a similar study, ephedrine was given to MG patients 50mg daily in 2 doses. When compared with the placebo group add- on ephedrine treatment increased QMG (Quantitative Myasthenia Gravis score) score by 1.0 points, which was significant for the group of trial patients as well as for the population treatment effect. It was also stated that ephedrine improved MG-composite scale by 2.7, MG activities of daily living profile by 1.0, and visual analog scale score for muscle strength by 1.1 [81].

5.4. Tubocurarine and Derivatives

Antinicotinic agents act on the nicotinic acetylcholine receptors. They prevent the transmission of signals from motor nerves to neuromuscular structures of the skeletal muscle. Nondepolarizing neuromuscular blocking agents prevent access of acetylcholine to the receptor, interrupting transmission at the skeletal neuromuscular junction without causing depolarization of the motor end-plate. They prevent acetylcholine from triggering muscle contraction and are used as muscle relaxants in convulsive states, as well as anesthesia adjuvants. The action of nondepolarizing drugs can be reversed by cholinesterase inhibitors like neostigmine [82,83].

Curare, the prototype of antinicotinic agents, is a common name for various South American poisons. It causes paralysis of mammalians by blocking transmission between nerve and muscle without affecting nerve conduction or muscle contraction directly. The main toxin of curare is the isoquinoline derivative D-tubocurarine, a classical example of a nondepolarizing agent that competitively blocks the action of acetylcholine on nicotinic receptors. Currently, D-tubocurarine is rarely used because much safer alternatives are available [84].

Nondepolarizing drugs can be divided into chemical groups of tetrahydroisoquinoline derivatives-tubocurarine itself, atracurium, doxacurium, mivacurium, and cisatracurium (Fig. 10).

Fig. (10).

Chemical structures of (D)-Tubocurarine, Atracurium besilate, Cisatracurium besilate, Mivacurium chloride, and Doxacurium chloride.

Tubocurarine, 7¢,12¢-dihydroxy-6,6¢-dimethoxy-2,2,2¢,2¢-tetramethyl tubocuraranium dichloride, is synthesized from an aqueous extract of the Chondodendron tomentosum, tetrahydroisoquinoline type alkaloid which contains isoquinaline subgroup and nitrogen is frequently in the quaternary form that increases its solubility. Its main bis quaternary skeleton with an approximate interonium distance of 10 nm was used as a template in the synthesis of semi- and synthetic compounds for the purpose of discovering better muscle relaxants rather than tubocurarine itself. Tubocurarine acts as a competitive inhibitor in the nicotinic acetylcholine receptor, meaning that the nerve impulse is blocked by this alkaloid.

Metocurine dimethyl-tubocurarine (Fig. 11) is a nondepolarizing neuromuscular blocker, a synthetic derivative of D-tubocurarine [85]. It is about twice as potent as D-tubocurarine and a quarter as potent as pancuronium. It has comparable onset and duration of action and speed of recovery to pancuronium and D-tubocurarine [86].

Fig. (11).

Chemical structure of metocurine.

Atracurium, a nondepolarizing neuromuscular blocking drug of the benzylisoquinolinium class, is another similar molecule to tubocurarine which is obtained via Hofmann degradation at physiological pH and body temperature. It is a competitive antagonist of the alpha subunit of the postsynaptic nicotinic receptor at the neuromuscular junction. It competes with acetylcholine for binding sites. Binding of the postsynaptic nicotinic receptor by atracurium prevents depolarization of the motor end-plate and subsequent skeletal muscle contraction. Unlike the binding of depolarizing agents, binding of atracurium or other nondepolarizing agents does not induce a receptor conformational change [87-89]. The obtained atracurium besylate is three times more potent than tubocurarine with lesser side effects, faster onset, and offset blockage than tubocurarine, and has less propensity than tubocurarine to release histamine.

Doxacurium is the benzylisoquinoline derivative with the longest duration of clinical activity. It is the most effective clinically obtainable neuromuscular blocking agent, with approximately twice the potency of pipecuronium or pancuronium. Doxacurium is a relatively new long-acting benzylisoquinoline compound that causes little to no histamine release or cardiovascular side effects [90].

Mivacurium is the only short-acting nondepolarizing neuromuscular blocking agent obtainable and metabolized by plasma esterases and presumably also in the liver. Mivacurium may also be potentially beneficial in patients with underlying neuromuscular disorders such as muscular dystrophy. In such patients, prolonged neuromuscular blockade may occur, resulting in even a single dose of an intermediate-acting agent such as atracurium, vecuronium. Consequently, the use of an agent with the shortest probable clinical duration may be helpful [91-93]. When compared with healthy control subjects, though there was no difference noted in the onset times, patients with Duchenne muscular dystrophy confirmed only a modest prolongation of the clinical effect of mivacurium [94].

The combination of the basic bis quaternary approach and bulky end groups separated by biodegradable ester linkages aid the synthesis of mivacurium and doxacurium. Mivacurium is shorter acting than atracurium and has no cardiovascular side effects. On the contrary, doxacurium is a long-acting drug, although it possesses two ester linkages in the structure.

5.5. Amaryllidaceae Alkaloids

One of the Amaryllidaceae alkaloids, galantamine [(4aS,6R,8aS)-5,6,9,10,11,12-hexahydro-3-methoxy-11-me- thyl-4aH-[1]-benzofuro[3a,3,2-ef][2]benzazepin-6-ol] (Fig. 12), was isolated from Galanthus L. and Leucojum L. species and also from Narcissus pseudonarcissus L. Galantamine is a competitive AChE inhibitor and an allosteric modulator of nAChR's, unlike other marketed drugs that inhibit AChE [95]. Galantamine easily crosses the blood-brain barrier and increases central cholinergic tone by inhibiting brain cholinesterases [96].

Fig. (12).

Chemical structure of galantamine.

Galantamine was evaluated for its activity on and BChE; it was found that galantamine is 50 fold selective to AChE. There was no difference between enzyme inhibition caused by galantamine between whole blood, plasma, and erythrocyte fractions; it is also concluded that galantamine does not accumulate in large quantities in red blood cells [97].

A study performed by Bickel et al. examined the pharmacokinetics, tissue distribution, and AChE inhibitory effects of galantamine in mice. Galantamine was applied at 4, 6, and 8 mg/kg (i.v). It has been reported that there is a rapid distribution of galantamine in tissues and red blood cells show a concentration 1.34-times greater than plasma. A maximum of 43% AchE inhibition was reported in brain homogenate [98].

With many studies, new analogues of galantamine have been synthesized, and higher efficacy was achieved on cholinesterase inhibition. 6-O-demethyl-6-O [(adamantan-1-yl)-carbonyl] galantamine hydrochloride (P11149) (Fig. 13) was found to have better oral therapeutic indices than galantamine and slower, lower, and more sustained maximal concentration levels [99].

Fig. (13).

Chemical structure of 6-O-demethyl-6-O[(adamantan-1-yl)-carbonyl]galantamine hydrochloride (P11149).

Bis-interacting ligands, different alkyl linkers (CH₂)_n with a terminal ammonium or phthalimido group were connected to the nitrogen of norgalantamine, and to the oxygen of 6-O-demethylgalantamine. Among the compounds studied, the most active ones were 9-Dehydro-10-N-demethyl-10-N-(80-phthalimidooctyl)-galanthaminium bromide and 9-Dehydro-10-N-demethyl-10-N-(100-phthalimidodecyl)-galanthaminium bromide (IC 50: 0.1x10^-7 M and 0.2x10^-7 M, respectively). It was reported that iminium function and N-alkylation of the nitrogen of the galantamine moiety with a phthalimido alkyl linker (n=8 or 10) increased AChE inhibition [100]. In addition, the alkylene component, when linked to galantamine increased AChE inhibition due to lipophilicity of alkyl and alkylene groups [101,102].

Galantamine, with a proven effect on the peripheral nervous system and central nervous system and less toxicity than pyridostigmine, is used in many countries of Eastern Europe for the treatment of myasthenia gravis and muscular dystrophy, residual poliomyelitis paralysis symptoms, trigeminal neurologia, and other forms of neuritides [103].

Lycorine, (1S,17S,18S,19S)-5,7-dioxa-12-azapentacyclo [10.6.1.0^2,10.0^4,8.0^15,19] nonadeca 2,4(8),9,15-tetraene-17,18-diol, is isolated from the Narcissus pseudonarcissus [104]. The first information on AChE inhibition of ungiminorine from the Lycorine series, with an, 86 μM IC50 value, reported by Ingkaninan et al. [105]. Lopez et al. reported that AChE inhibition with assoanin, oxoassoanin, pseudolycorine, and 2-acetoxypseudolycorine IC50 values were 3.87, 47.21, 152.32 μM, respectively. No specific effect of 2-acetoxypseudolycorine was observed on AChE [106]. Galantin (trimethoxy-substituted pseudolycorine) has moderate AChE activity (IC50 63.1), however, C-3/C-4 α-epoxide incartine showed no activity [107,108]. Narciprimine, the phenantridone alkaloid, is isolated from Cyrtanthus contractus N.E. Br. Although its structure is associated with many amaryllidaceae alkaloids, the characteristically amidic nature of nitrogen makes this compound different from the basic structure [109]. The AChE inhibition activity of narciprimine was surveyed in the experiment in which galantamine was used as standard. The inhibition potency of the galantamine (IC50 1.9 µM) was 40 times stronger than narciprimine (IC50 78.9 µM) [110]. The polyhydroxylated D-seco-lycorine analog narciclasine, and phenanthridone narciprimine exhibited moderate activity [110-112]. Interestingly strong AChE inhibition ability of 1-acetoxylycorine has been proved with an IC50 value of 0.96 μM. Lycorine and 1,2- diacetoxylycorine have lower activity and IC50 values were respectively found as 213 and 211 µM [113]. The chemical structures of the compounds are given in Fig. (14).

Fig. (14).

Chemical structures of lycorine, ungiminorine, assoanin, oxoassoanin, pseudolycorine, 2-acetoxypseudolycorine, galantin, incartine, narciprimine, narciclasine, 1-acetoxylycorine, 1,2- diacetoxylycorine.

5.6. Berberine

Berberine alkaloid (Fig. 15), which is found in several members of the Berberidaceae family, is effective on AChE inhibition [114, 115]. Xiang et al. explained that binding of the berberine and AChE is mostly directed by favorable entropy increase with a less impotent affinity. Berberine causes a decrease in enzymatic activity at a concentration well below the concentration that slowly exposes tryptophan residues to a more hydrophilic environment and releases the protein, indicating that the inhibition of AChE with berberine involves interference and minor conformational alteration of the protein [116].

Fig. (15).

Chemical structures of berberine, palmatine, and fangchinoline.

Concerning isoquinoline-derived alkaloids, these alkaloids design AChE ligands, as recent studies have indicated that the salts of berberine and tetrahydroprotoberberin are potent AChEIs [117, 118]. Huang et al. designed a study with new derivatives of berberine to examine their AChE inhibitory activity. Most of the derivatives reveal strong activity in the sub-micromolar range. The compound berberine linked with phenol by a 4-carbon spacer demonstrated the strongest inhibition of AChE with 0.097 μM IC50 value [119].

A number of active plant compositions can work synergistically, thus leading to a pharmacological action; therefore, combinations of berberine and other alkaloids have been studied for their AChE inhibitory activity. In a study conducted by Mak et al., Berberine and palmatine (Fig. 15) have been shown to synergistically inhibit acetylcholinesterase. The IC50 values were as follow: Berberine 0.52±0.042 μM, palmatine 0.46±0.013 μM and berberine +palmatine 0.17±0.023 μM [120]. More recently, Balkrishna and colleagues (2019) have shown that berberine, and palmatine have synergistic effects on acetylcholinesterase inhibition. Molecular docking studies have demonstrated that palmatine and berberine prefer to bind peripheral anionic sites, which may result in partial substrate blocking or blocking in product release [121].

Similarly, fangchinoline (Fig. 15) and berberine inhibited AChE in a dose-dependent manner. Different ratios (1:5, 1:2, 1:1, 2:1) of their combinations were tested in the study. IC50 values of combinations of 1:5, 1:2 and 2:1 were found to be 0.19 ± 0.02, 0.55 ± 0.17, and 0.79 ± 0.47 µM, respectively. It has also been shown that combinations are more active than the compounds alone [122].

5.7. Other Alkaloids with AChE Inhibitory Activity

Park et al. determined the inhibitory effect of Evodia rutaecarpa Bentham on in vitro acetylcholinesterase and its in vivo anti-amnesic effect. By sequential fractionation of E. rutaecarpa, the active ingredient has been shown to be dehydroevodiamine hydrochloride (Fig. 16). DHED inhibited acetylcholinesterase activity in a dose-dependent and non-competitive manner, and its IC50 value was determined to be 37.8 µM [123]. In a study investigating the structure-activity relationship for AChE inhibition, a benzene fragment was used instead of the indole fragment. Among the synthesized compounds, it has been determined that amidine structure compounds have moderate to strong AChE inhibitory properties [124].

Fig. (16).

Chemical structure of dehydroevodiamine hydrochloride.

A bioactive, 4,5-disubstituted N-methylimidazole, visoltricin generated by Fusarium tricinctum (Corda). In the study evaluating the anticholinesterase features and kinetic parameters of visoltricin (Fig. 17). N-methyl visoltricin showed low anticholinesterase activity in human serum, AChE, and BuChE with IC50 values 2.6 x 10^-4M, 4.0 x 10 ^-4 M, and 1.9 x 10 ^-4 M, respectively. Anticholinesterase activity of N-methyl visoltricin iodide was four times higher (7x 10^-5M). Kinetic studies showed a mixed type of inhibition for both compounds by partial binding of the inhibitor to the enzyme-substrate complex. It is stated that visoltricin is a reversible acetylcholinesterase inhibitor, and it is not hydrolyzed by the inhibited enzyme [125].

Fig. (17).

Chemical structure of visoltricin.

Pagliosa et al. evaluated the AChE inhibitory properties of galantamine, montanine, hippeastrine, and pretazettine (Fig. 18). Montanine inhibited > 50% of the enzyme at a dose of 1 mM. 30-45% AChE activity inhibition appointed at concentrations of 500 µM, and 100 µM, while galantamine revealed enzyme inhibition higher than 90% at 1mM, 500 µM and 100 µM concentrations. Hippeastrine and pretazettine showed no meaningful inhibition of AChE activity [126].

Fig. (18).

Chemical structures of montanine, hippeastrine, and pretazettine.

Karadsheh et al. reported inhibition of acetylcholinesterase (AChE) by caffeine, anabazine, methylpyrrolidine and various alkaloids, with physostigmine serving as a positive control. Caffeine, isocaffeine, morphine, myosmine,anabasine, nicotine, and 1-methyl-pyrrolidine were shown to be moderate inhibitors (Fig. 19). Caffeine was found to be the strongest AChE inhibitor (IC50 87 µM) in this group. 7,9-Dimethylxanthine 1-Methylpyrrolidinone, cotinine, and 1-Methylpyrrole showed low activity with IC50 values between 2056-18450 µM [127].

Fig. (19).

Chemical structures of caffeine, isocaffeine, morphine, myosmine, anabasine, nicotine, 1-methyl-pyrrolidine, 9-Dimethylxanthine 1-Methyl-2-pyrrolidinone, cotinine, and 1-Methylpyrrole.

CONCLUSION AND FUTURE PERSPECTIVES

Autoimmune disorders of the NMJ, even though infrequent, are a fascinating group of diseases, both scientifically and clinically. Although the pathophysiology of these conditions is now well understood, their clinical treatment is still largely based on anecdote and personal preference. Thus, a clear need for well planned randomized trials to explain the ideal treatment for these diseases is required. It is apparent that variability of plants and isolated compounds display therapeutic efficacy that is beneficial to the treatment of NMJ diseases. The majority of studies have focused on AChE inhibitors.

CONSENT FOR PUBLICATION

Not applicable.

FUNDING

MA was supported in part by grants from the National Institute of Environmental Health Science (NIEHS) R01ES07331 and R01ES10563.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.