Calpain Inhibition as a Magic Bullet
Info: 6657 words (27 pages) Dissertation
Published: 16th Dec 2019
Calpain Inhibition as a Magic Bullet
Abstract: Proteases are involved in a variety of diseases including the majority of neurodegenerative and neuromuscular disorders. The role of protease inhibitors as therapeutic agents has been the matter of intensive research.
Given the role of proteases in different disease states, it is appealing to develop a single agent as a “magic bullet” that would target all the disease states. Calpain inhibitors have been tested with success in animal models of human diseases.
In this review we summarize the associated neurodegenerative and neuromuscular diseases and the role of calpain in individual disorders. We describe the various instances in which a protease, in this case calpain, has been implicated. We discuss the results of calpain inhibition in a variety of pathological conditions and how targeted calpain inhibition could be beneficial. The focus of this review is to highlight the potential of targeted protease inhibitors as therapeutic agents in the treatment of neurodegenerative and neuromuscular disorders and to introduce three newer calpain inhibitors with improved characteristics.
Keywords: Calpain, Neurodegeneration, Calpain inhibitors, targeted-Calpain inhibition, Seizure, Parkinson’s disease, Huntington’s disease, Multiple Sclerosis
Calpain is one of the proteases that plays an important role in the life and death of mammalian cells including proliferation, differentiation and migration (1-3). Under physiological conditions, calpain is inhibited by its natural inhibitor calpastatin (4, 5). The rise in cytosolic free Ca2+ concentration following genetic defects, injury or inflammation activates the proteolytic enzyme by autocleavage of its subunits and degradation of its natural inhibitor calpastatin (6-9). The free enzyme then moves to the membrane and exerts its action (10-13). In its relation with cell death, calpain was primarily considered to be responsible for necrosis, but it is now well-known that calpain may play important roles in apoptosis as well (14). Although other proteases are also involved in muscle and neuronal degradation, calpain appears to play a major role in this process since neuronal protein degradation such as alpha-fodrin is significantly decreased when calpain is inhibited following central nervous system (CNS) injury (15-24). Thus, inhibition of calpain may help diminish the symptoms, or help treat these disease entities that result from abnormal activation and action of calpain.
Given the role of calpain in different pathological conditions, several calpain inhibitors have been developed and tested as potential therapeutic agents in animal models of diseases and in human trials (15, 19, 20, 22, 25-27).
Mechanisms of Neurodegeneration in Neurodegenerative and Neuromuscular Disorders
Different pathways lead to the abnormal and prolonged activation of calpain as a result of increased intracellular calcium. Of note, mechanisms other than abnormal calpain activation contribute to the pathophysiology of neurodegenerative diseases. Here we will simply discuss the role of calpain in the pathophysiology of individual diseases.
A. Alzheimer’s disease (AD)
Different hypotheses exist about the molecular mechanism of AD such as the cholinergic and the amyloid cascade hypothesis. In the amyloid cascade hypothesis the disease is initiated by the overproduction and extracellular deposition of amyloid β-peptide (Aβ) and intracellular deposition of neurofibrillary tangles (NFT). These depositions become the initiating factors for multiple neurotoxic pathways which may include excitotoxicity, Ca2+ homeostatic disruption, free radical production and inflammation in neurons. Upon binding with a putative membrane receptor, Aβ activates a molecular cascade that leads to activation of calpain and degradation of important proteins involved in synaptic plasticity and learning and memory (28). Among the consequences of this proteolysis, there is the decreased phosphorylation or increased degradation of the transcription factor cAMP response element-binding protein (CREB), resulting in a failure to maintain transcription, ultimately leading to synaptic dysfunction and cognitive abnormalities (29). Treatment with calpain inhibitor showed prevention of Aβ-induced dynamin 1 and tau cleavage in cultured hippocampal neurons giving further evidence for the role of calpain inhibition for potential treatment of AD (30).
A newer aspect of the disease pathology involves the activation of cyclin-dependent kinase (CDK) 5 by calpain which further strengthens the role of calpain in the disease pathology. Cyclin-dependent kinase 5 is a small protein serine/threonine kinase with close structural homology to the mitotic CDKs (1,2). Association of CDK5 with p35, its regulatory subunit, is critical for kinase activation. The p35/CDK5 kinase is required for neurite growth and lamination (31). To be activated, CDK5 has to be associated with its regulatory subunit, p35. It has been shown that various insults including ischemia, genetic alterations, excitotoxicity or changes in calcium ion homeostasis could cause the generation of p25 from p35, a truncated form. These factors activate calpain, which in turn processes the conversion of p35 to p25. The resulting truncated product accumulates in the neurons and activated CDK5 aberrantly, which results in deregulation and mislocalization of CDK5/p25 complex. This CDK5/p25 complex results in hyperphosphorylation of tau and causes disruption of the cytoskeletal elements, degeneration of neurons and apoptosis (32, 33). Treatment with calpain inhibitor resulted in decreased expression of calpain mediated p25 fragment (34). In addition, calpain inhibition improved memory loss in an animal model of AD by improving/reestablishing synaptic connection (29).
B. Amyotrophic lateral sclerosis (ALS)
The role of calpain in ALS has been emphasized and recent work has demonstrated the role of calpain in the pathophysiology of this disease (35, 36). Approximately 10% of ALS patients are familial cases and approximately 20% of which are caused by missense mutations in the enzyme Cu/Zn superoxide dismutase 1 (SOD1)(36) (37, 38).
Various mechanisms have been proposed to explain the loss of motor neurons in ALS, including oxidative damage, excitotoxicity from impaired clearing of glutamate, toxicity mediated by intracellular aggregates of mutant SOD1 and disruption of axonal transport caused by neurofilament disorganization (35, 36, 39).
There is growing evidence for involvement of members of the CDK family in neurodegenerative disorders and in the apoptotic death of neurons subjected to various insults, including ALS (40) (35, 39, 41)
It has been shown that blocking CDK5 activity protects cultured cortical and hippocampal neurons from fibrillary Aβ-mediated toxicity in vitro and in vivo as well. Evidence also indicates that lack of CDK5 activity results in degeneration of motor neurons (35, 40, 42). Appropriate control of CDK5 activity is crucial and loss of this control is detrimental to motor neurons. Calpain inhibition might be an alternative or even better approach in controlling the CDK5 activity and treatment of ALS.
C. Vestibulocochlear injury
Vestibulocochlear loss of structure and function, with symptoms of hearing loss, tinnitus, vertigo, pain, and neuropsychiatric disorders, can be caused by noise exposure, drug reactions and neurodegeneration and they can exist within the peripheral and central vestibulocochlear system(43). Loss of function in the cochlea can be caused by sound overstimulation, aging and side effect of drugs (44-46) and loss of spiral ganglion neurons (47).
The underlying pathophysiology is believed to be a disruption in the protein metabolism with resultant proteolysis. The neurochemical mechanisms of neuroprotection and apoptosis have been recommended for investigations of “ototoxicity” with calpain inhibitors (43).
Studies on the role of neurodegeneration in noise-induced hearing loss and tinnitus have been based on the hypothesis that tissue degeneration and inhibition of pathways leading to it form a potential for therapeutic intervention (48). The role of calpain in the pathophysiology of vestibulocochlear injury has been shown in different studies (48-51). In the cochlea, exogenous calpain inhibitors have been shown to protect hair cells against acoustic trauma (52, 53) and antibiotic ototoxicity (49). In addition, these inhibitors appeared to protect both the cochlear hair cells and ganglion neurons against hypoxia and neutrophin withdrawal (16). Long term safety of the calpain inhibitor leupeptin was established in guinea pigs by investigation of baseline and post treatment effects on cochlear blood flow, auditory sensitivity, and cochlear histology (50). The effects of sustained-release delivery of Leupeptin on the hearing of chinchillas produced no hearing loss at the early time points but did produce some hearing loss at later time points in 2 of the 5 animals. Application of Leupeptin to the middle ear cavity reduces hearing loss from exposure to impulse noise (51). Although calpain inhibitors are not strictly selective (54), these findings strongly suggest that calpain plays a role in hair cell and primary neuron degeneration in the damaged cochlea.
In the normal cochlea the presence of alpha fodrin has been demonstrated in hair cells, supporting cells, and neurons. In hair cells, alpha fodrin is concentrated in the cuticular plate and is seen along the lateral cell membrane (55). In support of this finding is the elevation of cytosolic Ca2+ level that occur in aminoglycoside-damaged hair cells, a condition that is required for calpain activation (1). In cochlear hair cells, typical signs of damage due to toxicity are alteration of the cell shape and disorganization of the cuticular plates. The degradation of fodrin by calpain in both cortical lattice and cuticular plates of the outer hair cells (OHCs) could account of such cell changes (56-58).
Excitotoxicity is involved in the pathophysiology and progression of several disorders of the central nervous system including epilepsy (59, 60). Neurodegeneration in the hippocampus and related memory impairments is observed both in patients and animal models of temporal lobe epilepsy (TLE) (61-63). Excitotoxicity is an overactivation of ionotropic glutamate receptors leading to an increase in the intracellular calcium concentration, triggering a cascade of cellular events resulting in calpain overactivation tissue damage (64-66). The hippocampus has highly enriched glutamatergic synapses and excitotoxic damage occurs following an excessive activation of glutamate receptors during epileptic phenomena (67, 68). Systemic injection of kainic acid (KA) (69) causes cell death in the hippocampus by excitotoxicity in a seizure-dependent process (64, 65, 70). Calpain is involved in the pathophysiology of seizure-dependent neurodegeneration (61, 63, 71-73). Seizures activity leads to calpain activation and proteolysis of spectrin as a result of KA injection in the hippocampus resulting in neurodegeneration in the hippocampus, (72, 74, 75). It is not clear if calpains are responsible for the neuronal death that follows convulsive activity in the KA model of epilepsy and whether calpain inhibition can reduce early neuronal damage. Thus, inhibition of calpain is a target for therapeutic approach of seizure-related neurodegeneration. Given the roles of pregabalin and calpain inhibition in the treatment of seizure–related neurodegeneration, combining the two drugs provides an excellent double therapeutic intervention for the treatment of seizure disorders.
E. Ischemic stroke
Interruption of blood flow to the brain causes membrane depolarization and disrupts Ca2+ homeostasis in neurons. The ischemic injury following reduced blood flow increases intraneuronal free calcium level leading to activation of calpain (54, 76-82).
Calpains and other proteases are known to be the major players in degradation of many key cellular proteins for mediation of apoptotic death of neurons in the lesion and the penumbra following brain ischemia as well as forcing the neurons to delayed death in ischemic penumbra (54, 80-84).
Mechanisms involved in neuronal injury after ischemia include highly reactive free radicals and cytotoxic cytokines which participate directly or in combination with calpain activation during brain ischemia. In vitro, nitric oxide (NO) regulates calpain and caspase-3 activation. Production of reactive oxygen species and other cytotoxic factors in the course of cerebral ischemia can prompt activation of the other Ca2+ independent proteases such as caspases (81). In addition, oxygen and glucose deprivation in brain slices caused calpain dependent conversion of the CDK5-activation leading to an increase in the enzymatic activity of the cofactor p35 to p25 (76, 85, 86). Overall, inappropriate imbalances between proteases and protease inhibitors in cerebral ischemia has led to extensive examination of calpain inhibitors as a therapeutic target for ischemic stroke (2, 66, 78, 86-89).
Using an animal model, multiple agents with calpain inhibition properties have been tested(90). Some experiments with calpain inhibitors have shown that administration of MDL 28,170 caused a dose-dependent reduction in infarct volume when administered 30 minutes after middle cerebral artery occlusion (MCAO). MDL 28,170 reduced infarct volume when therapy was delayed for 0.5, 3, 4, and 6 hours after the initiation of ischemia. The protective effect of MDL 28,170 was lost after an 8-hour delay (91). Rats treated with Cbz-Val-Phe-H showed significantly smaller infarct volumes. Intravenous injections of cumulative doses of 30 mg/kg or 60 mg/kg of Cbz-Val-Phe-H were effective in reducing infarction, edema, and calcium activated proteolysis(92).
The calpain inhibitor Calpeptin has shown improvement of neurological function, infarction volume and neuronal apoptosis of the CA1 sector of the hippocampus after focal cerebral ischemia reperfusion injury in rats. The results confirmed further the role of calpain inhibition in neuroprotection against focal cerebral ischemia-reperfusion injury(78). Pharmacological inhibition or conditional knock-out (CKO) of Cdk5 prevented neuronal death by blocking excitotoxicity in response to ischemia and Cdk5 CKO dramatically reduced infarctions following (MCAO) (76, 85, 86)
F. Huntington’s disease (HD)
Huntington’s disease is the most frequent of nine inherited neurodegenerative disorders caused by expansion of a CAG trinucleotide repeat encoding polyglutamine (polyGLYN). Activation of calpain and calpain cleavage products in the HD brains has been observed. These results suggest calpain cleavage of huntingtin plays a role in HD pathogenesis. Processing of mutant huntingtin (mhtt) is regarded as a key event in the pathogenesis of HD (93). Mutant huntingtin fragments containing a polyGLYN expansion from intracellular inclusions is more cytotoxic than full length mhtt. Exposure of primary neurons to glutamate or 3-nitropropionic acid (3-NP) causes degradation of wild-type huntingtin. In a nitropropionic acid-induced animal model of HD, activation of calpain was found to contribute to degeneration of striatal neurons (94).
The degradation process can be blocked by calcium chelators which causes reduction in the level of full-length wild-type huntingtin, leading to an increase in its cleavage products with a lower molecular mass. Mutant huntingtin is more easily cleaved by calpain, producing two huntingtin fragments. These fragments easily enter the nucleus to form intranuclear inclusions. Calpain-mediated cleavage of huntingtin mainly occurs in dying cells, suggesting the pathological processing of huntingtin in dying cells. Blockade of huntingtin cleavage reduces huntingtin aggregates and toxicity implying that calpain inhibitors may participate in the treatment regimen of HD.
G. Multiple sclerosis (MS)
All major myelin proteins are calpain substrates and calpain has been implicated in myelinolysis (13, 95-97). The role of calpain in the process of demyelination was suggested from the results of a study showing inhibition of myelin degradation by utilizing a thiol protease inhibitor E-64 on an extract of brain calpain (97).
In the white matter of MS patients it was found that calpain is significantly elevated (98) and increasingly expressed inside activated inflammatory cells following injury to the CNS (99, 100). Although other proteases are also involved in myelin degradation, calpain appears to play a major role in this process since neuronal protein degradation (alpha-fodrin and NFP) is significantly decreased when calpain is inhibited following CNS injury (15-24).
Demyelination leads to impairment or loss of axonal conduction (101, 102). In fact, in chronic forms of MS (103-107) and EAE (103) disease severity and the degree of permanent disability corresponds to the extent of axonal damage rather than myelin damage (108-110).
The exact mechanism of axonal injury is not fully understood. Several factors are believed to play a key role in the pathophysiology of axonal injury. Calpain is believed to play a major role in the pathophysiology of axonal injury particularly in the chronic phase of MS and EAE (111, 112). Demyelination results in impairment or loss of conduction of electrical signal which may be progressive. The neurons then try to compensate for the impaired electrical conduction by expressing more sodium channels (Nav1.6 channels) on the axons (102, 111, 113). The resulting increased influx of sodium into the axons via the Na+/Ca2+ exchanger (NCX) triggers increased Ca2+ influx which activates a number of enzyme cascades including calpain (111, 114). Inhibition of sodium channel using flecainide and phenytoin reduces axonal injury in EAE supporting the role of sodium channels in axonal pathology (3, 115).
H. Muscular dystrophy (MD)
Researchers have suggested that proteolysis by calpain causes muscle fiber degeneration in different muscular dystrophy and neuromuscular diseases such as Duchenne and Becker muscular dystrophies (DMD/BMD) as well as in inflammatory myopathy, acute quadriplegic myopathy, and distal myopathy (34, 116). It is thought that dystrophin deficiency results in membrane lesions causing leakage of intracellular constituents and influx of calcium ions. This influx of calcium ion has been proposed to account for the myofiber necrosis secondary to activation of calpain. The mechanism by which increased intracellular calcium in dystrophic myofibers causes muscle damage may be the activation of calcium dependent proteases, such as calpain (117-123). It is well known there is abnormal calcium accumulation in DMD myofibers as well as in murine MDX myofibers (118).
Further supporting the role of calpain in muscle injury, calpain inhibitors have been used to delay in muscle degeneration and necrosis in animal models of muscle injury and transgenic mice that overexpressed calpastatin showed reduction of dystrophic pathology (20, 25, 26, 124).
I. Parkinson’s disease (PD)
Parkinson’s disease is a progressive neurological disease characterized by loss of dopaminergic neurons in the substantia pars compacta (125). The molecular mechanisms mediating neurodegeneration of dopamine neurons in PD are poorly understood. Administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) increases calpain mediated proteolysis in nigral dopamine neurons in vivo (94).
Calpain expression and activity is elevated in postmortem PD brains (94, 126). In addition, the aggregation and accumulation of alpha-synuclein (α-syn) deposits leading to synaptic dysfunction and neuronal death is proposed to be one component in the pathophysiology of PD (28, 125).
One other mechanism proposed in the pathophysiology of PD as well as other neurodegenerative disease is the breakdown of CDK5/p35 into CDK5/p25 leading to increases in its kinase activity and neurotoxicity. Increased CDK5/p25 expression has been demonstrated in the brains of patients with Alzheimer’s and Parkinson’s diseases (127).
In 1-methyl-4-Phenylpyridinium ion (MPP+) treated granule neurons, calpain activation was determined showing an increase in calpain (74%) as measured by alpha-spectrin cleavage products. The results indicate that the cleavage is mediated by calpain. MPP+ prompted an increase in CDK5 expression, as well as the cleavage of p35-p25, in a time-dependent manner (128). Studies in rodent and cell culture models of PD suggest that treatment with calpain inhibitors (e.g., calpeptin, MDL-28170) can prevent neuronal death and restore functions. Inhibition of calpain using MDL-28170 or adenovirus-mediated overexpression of calpastatin, the endogenous calpain inhibitor, significantly attenuated loss of nigral dopamine neurons in an MPTP animal model (94). Our group has tested two calpain inhibitors in a small number of transgenic animal models of PD. Intraperitoneal administration of both calpain inhibitors, Gabadur and Neurodur for 30 days at 2mg/mouse/day in the PDGF α-Syn mouse model, is seen to have a dramatic effect on the deposition of α-Syn in examined brain tissue (hippocampus and cortex). Gabadur, decreases α-Syn deposition by approximately 60% and Neurodur, by approximately 40% in both regions. Calpain activity is decreased by approximately 70%. The animals tolerated the therapy without any observable toxic effects. Another hint for a role of calpain in PD is that overexpression of calpastatin leads to a reduction in truncated as well as aggregated α-Syn and of synaptic impairment in transgenic animal model of PD (129).
J. Traumatic Brain and spinal cord injuries (TBI and SCI)
Injuries to the brain or spinal cord trigger a process that results in neuronal damage in phases. The initial proposed mechanism after focal or global contusion appears to be excitotoxicity resulting in increased glutamate release and sustained overactivation of N-methyl-D-aspartate (NMDA) receptors resulting in increased Ca2+ overload as well as opening of voltage-dependent Ca2+ channels that are induced by depolarization immediately following CNS trauma (130, 131). Activation of the NMDA receptor results in a calcium influx into cells resulting in activation of the calcium dependent cysteine proteases including calpain which has been observed in models of excitotoxicity (132, 133), brain ischemia (2, 82, 84, 134, 135) and TBI models (136). In the secondary or delayed phase, tissue damage results from ischemia, loss of ion homeostasis, excitotoxicity, and free-radical production. TBI mediated axonal injury causes secondary damage to the myelin sheath and the myelin membrane of adjacent structures, instigating MBP degradation. This could initiate myelin sheath instability and demyelination, leading to axonal vulnerability and delayed neuronal injuries (137). The cleavage of cytoskeletal proteins also leads to axonal transport disruption and structural collapse, culminating in secondary axonal injury and possibly cell death. Among these mechanisms, excessive intracellular calcium Ca2+ accumulation and the activation of the neutral proteases, the calpains, seem to play the key roles (138-142).
After TBI in rats, a relative shift in μ-calpain activity from the cytosol to the total membrane fraction was observed from 3 to 24 h after injury, a time frame consistent with increased calpain autolysis and proteolytic activity (141, 143). This mu-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24-48 h after injury. The effect of mu-calpain activation on proteolysis of the neuronal cytoskeletal protein was evaluated using alpha-spectrin degradation products. Calpain expression in the cortex significantly changed during the time from TBI to death, and the most prominent expression was detected in the cortex 3 days after TBI. Immunoreactivity for alpha-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of alpha-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of alpha-spectrin breakdown products was observed between 24 and 48 h after injury (144).
The role of calpain in TBI and or SCI was tested using various calpain inhibitors, transgenic animals that overexpress the endogenous inhibitor calpastatin as well as calpain1 knockdown animal models. Pharmacologic calpain inhibition was found to be neuroprotective in both in vitro (145, 146) and in vivo models of excitotoxic injury (147) as well as in traumatic brain injury (148-150). In terms of pharmacological agents, various calpain inhibitors have been developed and tested (89, 149, 151-154).
Animals treated with the calpain inhibitor Calpeptin and methylprednisolone showed a decreased 68-kD neurofilament protein (NFP) degradation indicative of decreased calpain activity and reduction in apoptosis. Similarly, treatment by the calpain inhibitor E-64-d showed decreased degradation of the 68-kD neurofilament protein and internucleosomal DNA fragmentation in spinal cords of injured animals suggesting attenuated apoptosis in rat SCI as a result of decreased calpain activity (155, 156). Application of MDL28170 abated calpain I activation, inhibited apoptosis and neuron loss, quenched microglia and astrocyte activation, and significantly improved the neurologic deficit of locomotor function one week after spinal cord hemisection (157, 158). Intraspinal application of LV CAPN1shRNA 1 week before SCI in a Calpain 1 knockdown model demonstrated a significant improvement in locomotor function over 6 weeks after the initial injury when compared to LV control administration. In addition, calpain 1 protein levels were reduced by 54% 2 weeks after shRNA-mediated knockdown when compared to the LV control group, while calpain 2 levels were unchanged (159). The results from this experiment support the hypothesis that calpain 1 activation plays a role in tissue damage and impaired locomotor function and strengthens the potential of calpain inhibitors as therapeutic agents. Further supporting the role of calpain in TBI/SCI, Calpastatin overexpression significantly attenuated calpain-mediated proteolysis of these selected substrates acutely following severe controlled cortical impact injury (160).
Overview of Current Protease Inhibitors
Several attempts have been made to identify, develop and use calpain inhibitors (161, 162). The major difficulties in this matter have been calpain inhibitor specificity, water-solubility, cell membrane penetration and the ability to cross the blood-brain barrier (BBB) (13, 89). Over the years, some inhibitors have been developed that have better affinity and potency, but they still exhibit some of these limitations.
Current available protease inhibitors are broadly subdivided into 4 classes:
1. Peptidyl epoxides: irreversibly inhibit calpain and other cysteine proteases by forming an irreversible thioester bond with the active site cysteine (163-165). Its charged groups, however, prevent it from passing through the plasma membrane (e.g. E64).
2. Peptidyl aldehydes: form a reversible hemiacetal with the active site thiol. They suffer from poor solubility, poor membrane permeability and the inability to cross the BBB (e.g. Leupeptin).
3. Peptidyl ketomides: inhibit calpain reversibly and show improved cell and membrane permeability over the above mentioned groups (e.g. AK275 and AK295).
4. Non-peptide inhibitors: they target a site other than the active site and may show better specificity to calpain than other cysteine proteases. The inhibition mode is non-competitive and reversible. The binding of the inhibitor at an allosteric site may bring an alteration in the structure of calpain leading to an impaired confirmatory change and inactivation of the enzyme (e.g. PD150606 and PD151746).
Targeted Protease Inhibition
Our laboratory has been working on developing targeted protease inhibitors with improved characteristics for optimal effect. We have developed three protease inhibitors with different properties. Here, we describe the different protease inhibitors and their specificity with regard to organ systems/disease conditions.
- Gabadur (ALA 1.0)
Gabadur (ALA1.0) is synthesized by combining the anti-seizure/pain medication Pregabalin as a vehicle and Leupeptin as calpain inhibition. The concept was developed from the clinical observation of the efficacy of Gabapentin for a particular type of disabling subjective idiopathic tinnitus with early signs of neurodegeneration. Pregabalin is an analog of gamma-aminobutyric acid (GABA) and has been approved by FDA for use in neuropathic pain, adjunct therapy for partial seizure and as an anxiolytic agent (166). Gabadur’s structure is (3-aminomethyl-4amino-5methylhexanoic acid)-suc-leul-argininal. The pregabalin part of Gabadur acts on the voltage-gated calcium channel, thereby rendering its effect on the seizure activity. The leucyl-argininal part exerts its effect on calpain that is abnormally activated due to the excitotoxicity caused by the seizure. Pregabalin is a α2-δ ligand of the voltage-gated calcium channel similar to GABA (167-169). Pregabalin binding sites are upregulated in response to nerve damage (170), thus probably targeting the leucyl-argininal at the site of injury/insult. The combined effect is thought to minimize the the amount of the individual drugs needed and to reduce their side effects.
It has already been described in the literature that naturally occurring, carnitine, is distributed in muscle tissue at at least 100-200 times its concentration in plasma. We thus conceived the concept of covalently attaching carnitine to the active end of the Leupeptin molecule, leucyl-argininal allowing selective muscle absorption. The structure of Leupeptin, a well-known calpain inhibitor, and Myodur are identical at two positions, and it is the leucyl-argininal moiety there that is directly involved in calpain inhibition.
Leupeptin contains an acetylleucinyl group attached to the N-terminal leucyl and Myodur contains an aminocarnitine group attached to the amino group via a succinyl linker. Myodur targets the high affinity carnitine transporter, organic cation transporter (OCTN2) which is localized in the heart, the kidney and in skeletal muscle (171). It co-transports sodium and L-carnitine with high affinity along with drugs, and xenobiotics (e.g. choline, acetylcarnitine, quinidine, and verapamil) (172).This targeting principle results in enhanced potency and reduction in side effects when compared to Leupeptin alone. Myodur can be delivered to muscle tissue approximately 115 times more effectively than Leupeptin alone.
Taurine (2-aminoethanesulfonic acid) is a β-amino acid that relies upon a Na+-dependent transport system to pass through cell membranes (173). Two distinct high affinity Na+-dependent systems help transport Taurine. Taurine is synthesized in a limited amounts in the brain and significant amounts must be transported across the BBB (174). Due to structural similarity with taurine, cysteic acid (α-amino-β-sulfo-propionic acid) utilizes taurine transport to cross membranes (175, 176). Neurodur is synthesized by attaching the carboxyl group of cysteic acid to the leucyl-argininal of Leupeptin. This enables Neurodur to use taurine transporters to cross the BBB. Moreover, the carrier molecule taurine by itself has anti-inflammatory, calpain inhibitory and calpastatin upregulatory effects. This is an additional benefit of Neurodur. Limited experiments have shown the beneficial effect of taurine in closed head injury models in vivo (87, 177-181). The effect of Neurodur in suppressing inflammation, demyelination and protection of axonal injury in acute and chronic experimental autoimmune encephalomyelitis (EAE) has been shown in our previous works (27). The synergistic effect of Leupeptin and taurine make Neurodur a very appealing therapeutic agent in treating TBI patients.
All forms of tissue breakdown are regulated by protein synthesis and protein degradation. When protein degradation exceeds the synthesis pathway atrophy occurs in all forms of tissue wasting. This is most easily observed in muscle as it makes up approximately 40%-50% of the mass of an animal (182-195). Muscle wasting can also be the result of neuromuscular junction dysfunction or neuronal injury in which proteases play a role (196, 197). Thus, considering the role of protease inhibition has been reasonable in treating diseases with extensive tissue wasting. With the discovery of calpain (198) and the role of calpain in different disease conditions, the focused attention on inhibition of this protease began. The initial studies using Leupeptin, a thiol protease inhibitor discovered by the Umezawa group, showed promising results (199, 200). The early studies in muscle cultures incubated with Leupeptin showed a remarkable extension of the fiber lifespan and robustness of the fibers. These studies were sufficiently encouraging to enter into a series of in vivo animal model studies including denervation atrophy, the MDX genetic mouse models of DMD and EAE, an animal model of MS (26, 27, 201). An early study of denervated chicken pectoral muscle showed that the calpain inhibitor Leupeptin, when injected intramuscularly into the denervated pectoral muscle decreased the muscle atrophy by approximately 45 to 48%(202). This in vivo demonstration of the therapeutic effectiveness of calpain inhibitor was extremely encouraging and led to an extensive in vivo study in monkeys using Leupeptin to prevent denervation atrophy (19-21, 25-27, 201). Most interesting was the observation that calpain activity did not return to zero, but to close to control values depending upon the doses used suggesting that calpain inhibitors act in a manner to replace the endogenous inhibitor of calpain, calpastatin.
Similar studies with Leupeptin were carried out in an MDX mouse model of human DMD. In this study, although untreated muscle leads to loss in muscle fiber size calpain inhibition can prevent the decrease in myofiber diameter in both the gastrocnemius and diaphragm muscle of the MDX mouse after 60 days of treatment with Leupeptin (26).
Another area of calpain involvement is MS. Leupeptin has been used for the treatment of EAE. Unfortunately Leupeptin does not cross the BBB and Leupeptin has to be directly injected in to the CNS (203) or liposome-encapsulated in order to cross the BBB (22). The disadvantage of these ways of administering Leupeptin is the invasiveness of the intrathecal application and the degradation of the liposome-encapsulated drug when administered orally.
It became apparent that attaching the protease inhibitor to a carrier for selective delivery would have significant advantages. Targeted inhibition allows dose reduction of the individual agent and results in fewer side effects. It also increases the amount of drug available at the desired site of action. The advantages of targeted protease inhibitors are numerous, including:
- Delivery of the drug to the area of calpain overactivation by concentrating the amount of inhibitor in the area of the interest and reducing the amount of calpain inhibition in different organs/ organ systems where calpain is physiologically active.
- Reduction of adverse effects of the drug and toxicity by using a smaller dose.
- Increasing the window of opportunity in treating diseases since calpain is involved in the late stages of the disease.
Earlier studies from our laboratory using Leupeptin showed delay of muscle degeneration and necrosis in mdx mice and recovery of neuromuscular segment after peripheral nerve injury (19, 20, 25, 26). Our multiple sclerosis project showed suppression of inflammation and demyelination along with prevention of axonal injury using Neurodur (27). In a series of experiments we have shown a dose-dependent inhibition of calpain and suppression of demyelination and inflammation. In another experiment using an animal model of retinal ischemia, we found that post ischemic administration of 20 mg/kg of CYLA provides significant retinal preservation (204).
Preliminary results from an animal model of Huntington’s disease (3-nitropropionic acid induced striatal damage) using Gabadur showed about 50% preservation of the striatal structure. Western blot analysis using alpha fodrin antibody showed suppression of the full-length fodrin proteolysis in the Gabadur treated groups. Partial results from this experiment were presented at the Experimental Biology (EB) scientific meeting in April, 2009. The preliminary results from another project using an animal model of Parkinson’s disease showed reduction of α-Syn by Neurodur (40%) and Gabadur (60%). This model gives the strongest indirect evidence that the protease inhibitors can cross the intact BBB as compared to (as well as?)disease disrupted BBB. Currently experiments are being conducted in animal models of noise induced sensorineural hearing tinnitus as well as traumatic brain injury using our targeted calpain inhibitors.
The works described above show diverse involvement of calpain in the wide variety of disorders; thus the genesis of the title and the use of the term “magic bullet”.
With calpain as a “target” we have designed a potential “magic bullet” inhibitor of calpain and have shown its effect in animal models of multiple neuromuscular and neurodegenerative disorders. Thus it is not unrealistic to consider that Ehrlich’s dream of a “magic bullet” to treat a class of diseases pharmacologically may be more a reality than previously imagined.
Given the role of calpain in the pathophysiology of numerous disease states, including various neurodegenerative and neuromuscular diseases, and especially considering that their involvement in delayed tissue destruction allows an increased window of opportunity for treatment, calpain inhibitors either as a single agent or in combination with other pharmaceutical agents are appealing new therapeutic agents.
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