Fasudil And Its Analogues Biology Essay

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Rho kinase plays a critical role in actin cytoskeleton organization and is involved in diverse fundamental cellular functions such as contraction and gene expression. Fasudil, a ROCK inhibitor, has been clinically applied since 1995 for the treatment of subarachnoid hemorrhage in Japan. Increasing evidences indicate that fasudil could exhibit markedly therapeutic effect on central nervous system (CNS) disorders, such as Alzheimer's disease.

Areas covered- This review summarizes results from supporting evidence for the potential therapy for fasudil against a variety of CNS diseases. And the properties of its analogues are also summarized.

Expert opinion- Current therapies against CNS disorders are only able to attenuate the symptoms and fail in delaying or preventing disease progression and new approaches with disease-modifying activity are desperately needed. The dramatic effects of fasudil in animal models and/or clinical applications of CNS disorders make it a promising strategy to overcome CNS disorders in human beings. Given the complex of CNS disorders, further efforts are necessary to develop multifunctional fasudil derivatives or combination strategies with other dugs in order to exert more powerful effects with minimized adverse effects in the combat of CNS disorders.

Key words: Rho kinase; fasudil; central nervous system disorders; analogues

Alzheimer's disease (AD) Parkinson's disease (PD), spinal cord injury (SCI) and stroke, are common CNS disorders characterized by neuronal network deterioration [1]. The market value for AD and PD treatment exceeded $6.5 billion in 2009, and will surpass cancer as the second cause of death in the elderly [2]. However, current therapies against these disorders are only able to attenuate the syptoms and fail in delaying or preventing disease progression and new approaches with disease-modifying activity are desperately needed. Rho/ ROCK, which is involved in a wide range of patho-physiological changes in the actin cytoskeleton, including bone metabolism and cell adhesion [3-6], has been shown abnormal activation in a number of CNS disorders including SAH, AD and MS [7-10]. This in turn renders inhibition of Rho/ROCK as a hopeful weapon against CNS disorders. Fasudil, the only clinically available ROCK inhibitor, has numerous beneficial effects including vascular dilation, neuroprotection, and promotion of axonal regeneration, which providing new insights into the treatment for CNS disorders. In this review, we summarized that the Rho/ROCK pathway and the potential of fasudil in the treatment for CNS disorders as well as the study of its analogues. Moreover, the combination or multi-functional strategies of fasudil were also discussed.

1. Rho and ROCK

The Rho family of small GTPase, including Rho, Rac, and Cdc42, is a subfamily of the Ras superfamily [11, 12]. As other Rho-GTPases, Rho acts as a molecular device that cycles between GDP-bound (inactive form) and GTP-bound (active form) conformation. Guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP to activate RhoA while GTPase-activating proteins (GAPs) inactivate RhoA by stimulating the intrinsic GTPase activity [13]. The active form, Rho-GTPase, regulates cell shape, motility, proliferation, and apoptosis [14-16]. The downstream targets of Rho include p140mDia, p21-activated protein kinase (PAK), protein kinase N (PKN), rhophillin, and so on [17].

Rho-kinase (Rho-associated coiled-coil-containing protein kinase, ROCK), a member of the AGC (PKA/PKG/PKC) family, is one of the best-characterized effectors of small GTPase RhoA [18, 19]. Activated RhoA directly interacts with the C-terminal portion of the coiled-coil domain of ROCK and causes a conformational change, leading to ROCK activation. The activity of ROCK can also be modulated through interacting with the C-terminal pleckstrin-homology (PH) domain with lipid mediators (such as sphingosylphosphorylcholine), mechanical stress and proteolytic cleavage of its inhibitory C-terminal domain by caspase-3 [20, 21]. ROCK have been proved to phosphorylate various downstream substrates, including the myosin binding subunit of myosin light chain (MLC), LIM kinases, ezrin/radixin/moesin (ERM) and adducin [22, 23], which enables ROCK to modulate actin cytoskeleton organization, stress fiber formation and smooth muscle cell contraction [18, 22-26].

Two members of ROCK family, ROCK1 and ROCK2 were confirmed till now [27], Both of them are constitute of an amino-terminal kinase domain, a Rho-binding domain (RBD) which is situated within the mid-coiled-coil-forming domain, and a pleckstrin-homology domain containing a carboxy terminal cysteine-rich domain (CRD) (see Figure 1A). The two isoforms share an overall sequence similarity at the amino-acid level of 65% and in their kinase domains of 92% [28, 29]. Despite the striking similarity of the protein sequences of the two ROCK isoforms, it has been reported that there are significant differences regarding of their tissue distribution [30]. ROCK2 is mainly expressed in brain and skeletal muscle, while ROCK1 is prominent in liver, testes and kidney. By using gene knock-out technique, different phenotypes were found in ROCK1-knockout (ROCK1-/-) and ROCK2-knockout (ROCK2-/-) mice [31-35]. The two types of mice exhibit different phenotype under different genetic backgrounds (reviewed in Shi J and Lei W [36]). Interestingly, ROCK1-/- and ROCK2-/- mice develop normally and are apparently healthy and fertile after surviving from their intrauterine and perinatal problems [36]. Moreover, no compensatory up-regulation of the ROCK1 is observed in ROCK2-/- mice and vice versa [36].

2. Development of ROCK inhibitors

Basing on these studies carried on animals, over-expression of ROCK is involved in the pathogenesis of cardiovascular and cerebrovascular diseases and ROCK inhibitors show dramatically beneficial effects in animal diseases' models [37]. Consequently,an increasing amount of effort has been targeted to the research on the development of ROCK inhibitors [37].

Isoquinoline derivatives, especially fasudil, are typical ROCK inhibitors. Hydroxyfasudil is an active metabolite of fasudil in vivo, which has higher affinity to ROCK than the latter [7]. Another isoquinoline derivative, H-1152P, is optimized on the basis of fasudil. Through competitively binding to the ATP binding pocket, Y-27632, another type of ROCK inhibitor, inhibits both ROCK1 and ROCK2. Additionally, Y-27632 also inhibits PKA, PKC and citron kinase. Optimization of these compounds leads to a more potent ROCK inhibitor, Y-39983, which is benefit for the treatment of the glaucoma [7].

The inhibitors mentioned above are the most commonly used pharmacological ROCK inhibitors that target ATP-dependent kinase domain and are equipotent in regarding of ROCK1 and ROCK2. In addition, these inhibitors have possible non-selective effects and also inhibit other serine/threonine kinases including PKA, PKG and PKC at higher concentrations [37]. More potent and more selective ROCK inhibitors are needed urgently to realize efficient treatment with minimized adverse effects. Encouragingly, a ROCK2 specific inhibitor, SLx-2119 recently has been developed [38].

3. A summary of fasudil

Fasudil (hexahydro-l-(5-isoquinolylsulfonyl)-1H-1, 4-di-azepime), also named as HA-1077, is a novel isoquinoline sulfonamide derivative and the only clinically available ROCK inhibitor co-developed by Asahi Kasei of Japan and Department of Pharmacology of Nagoya University. Fasudil, which is mainly distributed in the stomach and intestine [39-41], is water soluble and orally effective. It has a short half-life (t1/2=0.5 h) and could be converted into a more active metabolite, hydroxyfasudil in vivo [42]. The latter one also has a longer half time (t1/2=2.9h) and mainly distributed in liver and kidney [42]. Both of Fasudil and hydroxyfasudil have low brain penetration ability [39], and liposome preparation[39] or myelin injection was applied [43] to improve its efficacy and to reduce the adverse effects. Fasudil inhibits the phosphorylation of MLCK [23], up-regulates endothelial nitric oxide synthase (eNOS) expression, decreases smooth muscle spasm and exerts significant cerebral vessels dilation, via blocking intracellular calcium channels (rather than extracellular calcium ion) [44, 45]. It can also inhibit the migration of inflammatory cells and increase the expression of eNOS [46]. Clinical studies showed that it has therapeutic effect on angina, hypertension, coronary vasospasm and coronary recanalization after operation of restenosis and atherosclerosis with favorable prognosis and minor side effects [37]. Most studies to date showed beneficial effects of fasudil on animal models of CNS disorders. In this article, effects of fasudil on several CNS disorders, such as SAH, cerebral stroke, AD and MS were summarized as follows.

4. Effects of fasudil on CNS disorders

It has been reported that Rho/ROCK pathway is closely associated with the pathological process of CNS disorders, including AD and cerebral stroke. Therefore, Rho/ROCK pathway is becoming a vital target in treating CNS disorders. And the only clinically available ROCK inhibitor, fasudil could [9, 47-50]: 1) suppress tissue factors induced by tumor necrosis factor alpha (TNF-α) in vascular endothelial cells; 2) activate endogenous neural stem cells of CNS; 3) increase astroglial cell stimulating factor; 4) inhibit intracellular calcium release; 5) dilate brain blood vessels; 6) protect nerve cells; 7) improve the nerve function; and 8) promote axonal regeneration, thus making it a potential therapeutic indication for many CNS disorders, such as SAH, AD and MS.

4.1 Subarachnoid hemorrhage (SAH)

SAH results from head trauma or spontaneously from the rupture of cerebral aneurysms [51]. And a SAH-induced cerebral vasospasm, characterized by increased constriction of cerebral arteries, results in tissue damage, stroke, and even death. It is reported that fasudil is no less effective than nimodipine, a L-type voltage gated calcium channels blocker [52], for the mitigation of cerebral vasospasm and the following ischemic injury in patients undergoing operation for SAH (fasudil: 10 mg/d, nimodipine: 1 mg/d, taking immediately after surgery for 14 d) [52]. Recent studies have proved that fasudil show better effects than nimodipine [53]. More importantly, post-marketing surveillance studies on SAH patients have shown that fasudil was well tolerated and safe in over 1400 patients examined [54]. Additionally, 10 patients received selective fasudil (a microcatheter inserted in intracranial arteries) were more beneficial than 10 other patients who received nonselective fasudil (a microcatheter inserted in the cervical arteries) [55]. Fasudil is beneficial for cerebral vasospasm through inhibiting ROCK and MLCP, decreasing smooth muscle spasm, attenuating myosin phosphorylation, and significantly reducing the blood viscosity [56], protects neurons via inhibiting of glutamate-induced excitotoxicity and the release of intracellular Ca2+ in ischemic area, and significantly dilates cerebral vessels by up-regulating eNOS expression and subsequently increasing NO production [55].

4.2 Cerebral stroke

Ischemic stroke, for example cerebral infarction, is one of the most common CNS disorders [57]. Cerebral infarction is characterized by high death rate and high disability rate. Accumulating evidence showed that fasudil could overcome this kind of ischemic brain injury. Some researchers argued that the beneficial effect of fasudil is associated with blocking ROCK, increasing NOS expression and cerebral blood flow preserving endothelial function and ameliorating leukocyte trafficking in the microcirculation [58]. Noteworthy, the delayed treatment of fasudil is still able to prevent neuron death from ischemia, implicating that fasudil has a wide therapeutic window for cerebral stroke. In a clinical trial carried on 160 patients, who took fasudil in 48 hours after ischemic stroke attack (i.v. 60 mg fasudil twice per day, 14 days), showed that fasudil obviously enhanced the nerve system function without severe side effects [28].

4.3 Spinal cord injury (SCI)

Although the pathogenesis of SCI is able to be explained at the molecular level, it is still a threatening disease to human beings [59, 60]. Indeed, success in an animal model has led to only one proven, though controversial, clinical intervention, namely methylprednisolone. Spinal cord contusion was induced in rats by applying an aneurysm clip extradurally to the spinal cord at T-3 for 1 min. In fasudil-treated group (i.p. 10 mg/kg), significant improvement in behavioral score was demonstrated, whereas in methylprednisolone-treated group, no beneficial effects were shown [61]. In another experiment carried on Japanese white rabbits, fasudil (infused into the isolated segmental lumbar arteries, 0.1 mg/kg) showed neuroprotective effects against ischemic spinal cord injury by reducing the number of infiltrating cells and elongating the expression of eNOS [62]. Moreover, fasudil is reported to significantly decrease inflammasome activation, pro-inflammatory cytokines such as tumor necrosis factor (TNF-α) and interleuchin-1β (IL-1β) production [63]. In other studies, fasudil not only enhanced nerve-fiber growth beyond the lesion site, but was also neuroprotective and could decrease tissue damage and cavity formation [28, 64]. Moreover, fasudil could normalize spinal blood flow due to its vasodilatory effects, thereby further enhance tissue preservation [59]. After comprehensive analysis, it is obviously that fasudil, with more powerful therapeutic effect on SCI than methylprednisolone, may represent a useful therapeutic perspective in the treatment of SCI.

4.4 Alzheimer's disease (AD)

AD, a progressive neurodegenerative disease, is pathologically characterized by intracellular neurofibrillary tangles and extracellular amyloid aggregates. However, no effective treatment for AD is currently available [65]. Neurofibrillary tangles contain aberrantly phosphorylated tau protein whereas the amyloid aggregates are formed primarily by toxic 42-amino-acid long amyloid-β (Aβ) peptide [66]. A large number of data from study of human genomics indicates a close relationship between Aβ42 and the pathology of AD. Also, correlation between decline of cognitive ability and the oligomer of Aβ42 has been reported [67]. Therefore, inhibition of Aβ42 can be considered an effective treatment for AD. One of the reported means to repress Aβ42 is to restrain the Rho-ROCK pathway. After intra-cerebroventricular injection (i.c.v.) of fasudil, both of the experiments carried in vitro and in PDAPP transgenic mice indicated that Aβ42 rather than the overall Aβ level has been reduced. Fasudil can also promote the regeneration of neuron and repair the neural circuits damaged by Aβ [28]. Another research pointed out that an evident growth in length and lessen of branching of the dendrite of pyramidal cells in hippocampal CA1 region after treating APP/PS1 transgenic mice with fasudil 24-26 d (i.c.v., 0.6 mg/kg/d) [10]. Recent study showed that the learning dysfunction in rats caused by i.c.v. of streptozocin, a common used model of sporadic AD could be reversed by fasudil (i.p. 10 mg/kg, for 4 weeks), suggesting that fasudil has potential anti-dementia properties [67]. In conclusion, fasudil might be a practical treatment of AD [37].

4.5 Parkinson's Disease (PD)

PD, a common neurodegenerative disorder, affects 1.5% of the global population over 65 years of age [68]. Axonal degeneration is one of the earliest features of PD pathology and inhibition of axonal degeneration thus become a pivotal target in PD treatment [69]. In the in vitro 1-methyl-4-phenylpyridinium cell model and in the sub-chronic in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, treatment of fasudil (oral gavage, 30 mg/kg) resulted in a overt improvement of dopaminergic cell loss in both models. In addition, doparminergic terminals were preserved and the motor performance was clearly improved after fasudil application. In this study, the AKT pathway was supposed to be a key molecular mediator for neuroprotective effects of ROCK inhibition [69]. Another ROCK inhibitor Y-27632 also showed beneficial effects in mice treated with MPTP [70]. These results demonstrated that Rho/ROCK pathway is involved in the MPTP-induced doparminergic degeneration and inhibition ROCK may provide new neuroprotective strategies against progression of PD.

4.6 Neuropathic pain

Neuronal injuries in the peripheral nervous system (PNS; peripheral nerves, dorsal

root ganglia (DRG) and dorsal roots) or in the CNS (thalamus or spinal cord) of human beings can lead to a chronic pain state known as neuropathic pain [71]. The relationship between ROCK and pain has been rarely reported. A small number of studies show that Y-27632 could reduce the pain induced by lysophosphatidic acid [72]. The effects of fasudil in different preclinical models of neuropathic, osteoarthritic (OA), and inflammatory pain as well as capsaicin-induced acute pain and secondary mechanical hypersensitivity were evaluated [73]: fasudil at the highest dose tested (30 mg/kg) significantly mitigated mechanical allodynia in spinal-nerve ligation (SNL; 77%), chronic constriction injury (CCI; 53%), capsaicin-induced secondary mechanical hypersensitivity (63%), sodium iodoacetate-induced OA pain (88%), and capsaicin-induced acute flinching behaviors (56%). However, fasudil (at 30 mg/kg) failed to produce significant effects on inflammatory thermal hyperalgesia after carrageen an injection and mechanical allodynia after Complete Freund's Adjuvant (CFA) injection. Moreover, treatment of fasudil (i.v., 10 mg/kg) significantly reduced both spontaneous and evoked firing of wide dynamic range (WDR) neurons in SNL, but not in sham rats, suggesting that the acute administration of fasudil produces efficacy in both neuropathic and nociceptive pain states at doses devoid of locomotor side effects, with specific effects on WDR neurons [73]. One of the fasudil analogues, H-1152P, relieved neuropathic pain in an L5 spinal-nerve-transection model [74]. These results show that ROCK is an important target responsible for the induction and also maintenance of persistent pain states, and fasudil might have therapeutic effects on both neuropathic and nociceptive pain.

4.7 Experimental autoimmune encephalomyelitis (EAE)

MS is a chronic neurological disease with onset primarily between the ages of 20 and 45 that may lead to different degrees of disability [75]. It is an inflammatory demyelinating disease of CNS, in which an autoimmune attack is supposed to be mediated by myelin antigen-specific Th1 cells. EAE is a prototype animal model of a Th1 cell-mediated CNS demyelinating disease that shares some clinical and pathological features of MS. Increasing evidence suggests that demyelination is related to the recruitment of Th1 cells and macrophages into the CNS, accompanied by microglia activation. The inflammatory cascade stimulated by these inflammatory cells ultimately leads to neuroinflammatory injury and myelin sheath destruction [76]. Yu J et al,.[77] found that ROCK inhibitor could hamper the inflammatory cells from penetrating the brain endothelial cells, implicating that ROCK is required during the migration of inflammatory cells. Fasudil (i.p., 5 mg/kg) was able to inhibit the up-regulation of ROCK2 in EAE rat's spinal cord and brain peripheral vascular clearance, down-regulate of IL-17, overtly reduce the expression of IFN-γ, inhibit T cell proliferation and prevent the inflammatory cells into the CNS. The mechanisms underlying the amelioration in EAE caused by fasudil may also include inhibiting TLR-4, p-NF-kB/p65, and inflammatory cytokines (IL-1b, IL-6, and TNF-a) and enhancing IL-10 production in spinal cords [78]. Inhibition of ROCK using fasudil may be a promising new therapeutic strategy for MS [79].

4.8 Epilepsy

Epilepsy, which is characterized by recurrent and unpredictable interruptions of normal brain function, is a kind of brain disorder [80]. Epilepsy is various disorders reflecting underlying brain dysfunction that may result from multiple causes rather than a singular disease entity [81]. The excessive enhancement of excitatory neurotransmission and/or the reduction of inhibitory pathways, as well as the regulation of some signal transductions may cause seizures. SY Inan et al.,[82] evaluated the function of ROCK inhibitor fasudil in three models of epilepsy: Pentylendetetrazole (PTZ), maximal electroconvulsive shock (MES), and PTZ kindling epilepsy. The results showed that, in the MES model, fasudil (i.p., 25 mg/kg 30 min before electric shock) suppressed the percentage of tonic convulsion index and recovery latency for righting reflex in the mice excited with MES. In the case of PTZ model, fasudil diminished onset of myoclonic jerks, clonic convulsions and tonic hindlimb extensions. Repeated medication of another ROCK inhibitor Y-27632 could prevent the development of PTZ kindled epilepsy by reducing the average seizure levels. They also found that, by the PTZ chronic sub convulsive dosage, the translocation of Rho to the plasma membrane increased, indicating that the Rho/ROCK pathway was activated in the epileptic seizures. These findings suggested that the Rho/Rho-kinase signaling may play a pivotal role in epilepsy induced by PTZ and MES. ROCK inhibitors are hopeful to become a new antiepileptic drug.

Up to date, fasudil has also been reported to be a new promising therapeutic strategy for many CNS disorders, such as SAH, AD and MS. The observations mentioned above raise the possibility that Rho/ROCK pathway plays a critical role in the pathology of CNS disorders. Although demonstrated to exhibit certain therapeutic effects in a variety of animal models of CNS disorders (Table 1), it still needs further safety and efficacy assessment whether fasudil could be put into clinical use.

5. Fasudil analogues

Fasudil competitively binds with the ATP binding sites of Rho kinase catalytic domain, and has the equal blocking potency to ROCK1 and ROCK2. The amino acid sequences in the ATP binding site region of protein kinases are highly homologous and therefore its selectivity for ROCK is limited [4]. In order to yield highly specific ROCK inhibitors, several fasudil analogues were synthesized.

The structural optimization of fasudil is mainly through the modification of three parts of fasudil, namely homopiperazine diamine, isoquinoline ring and sulfonyl connection group (Figure 2). A series of fasudil analogues were synthesized and their selectivity and inhibitory activity against ROCK were evaluated [16,48,76-82].

5.1 Modification of homopiperazine ring

The kinase inhibition properties of six analogues of fasudil in figure 3 were evaluated respectively. 1, containing no methyl substitution on the hexahydro-1H-1, 4-diazepine region, showed broad specificity. H-1152P (2) produced the most potent activity against ROCK2 in this series of analogs, as anticipated [83-85]. Rho-kinase inhibition became weaker when the 3-, 5- or 6-position was methylated (3, 4 and 5). The methylation at the 2-position of the hexahydro-1H-1,4-diazepine moiety is preferable for ROCK specificity since inhibitory activity against ROCK reduces as the 2-positioned substituent of hexahydro-1H-1, 4-diazepine was increased [86].

According to Breitenlechner et al. [83], there were some interactions between PKA and fasudil, and PKA inhibition by fasudil could be reduced by blocking the 4-amino group of hexahydro-1H-1, 4-diazepine. However, such an arrangement occasionally totally inactivates the inhibitor. Tamura et al.,[86] pointed out that acetyl derivative of the H-1152P analogue lost inhibition activities, indicating that the amine is important for the kinase inhibitors. The acylation of amino on the Homopiperazine ring appears to be a possible solution not only to retain the amino and also to reduce hydrogen bond forming between the amino and PKA. Tamura et al.,[86] synthesized glycine derivatives, compound 7, 8 and 9, confirming that ROCK is able to accept the molecules of acylating amino, thus being blocked [86].

5.2 Modification of the isoquinoline ring

Introduction of different groups to the 2-position of isoquinoline ring changed the activity of fasudil. The introduction of methyl, namely H-1152P, leads to higher selectivity and inhibition properties compared with fasudil, whereas the introduction of ethyl cyanoacrylate, hydroxyl results in lower selectivity and lower inhibition potencies. Interestingly, the activity was increased as larger halogens substituted the methyl group. A chloro-analogue of fasudil has similar inhibition activity against ROCK as H-1152P. However, inhibition against ROCK decreased as the halogen atom was enlarged. The substitution of ethenyl group leads to a potent compound 15, which possessed more potent and specific inhibition against ROCK. These results suggested that both potency and specificity of the chemicals against ROCK were improved by replacing the methyl group with ethenyl group [48].

5.3 Modification of the para- sulfonyl

Sulfonyl substituted by their electronic isostere such as carbonyl was found to be of no activity towards ROCK, suggesting the sulfonyl substitute is necessary for the inhibition of fasudil against ROCK [16].

5.4 Other analogues

Peter Ray et al.,[87] obtained 16 and its derivative 17 using the method of fragment-based drug design. 16 was equipotent against both ROCK1 and ROCK2, showed good in vivo efficacy in the spontaneous hypertensive rat model, and was less selective than hydroxyfasudil for the AGC-related kinases, in particular PKA and PKC. In contrast, 17 had poor bioavailability although its affinity and potency towards ROCK had been improved. In order to maintain the pharmacokinetic properties of 16 and to enhance its potency and selectivity, Peter et al., synthesized 18, of which inhibitory effect against ROCK is 10 times than that of hydroxyl fasudil, and of which selectivity is also better than that of hydroxyl fasudil. In addition, 16 pronouncedly reduced the blood pressure in spontaneously hypertensive rats, and showed stronger efficacy than that of other Y-27632 analogues such as 19-21. Analogue 16 has strong inhibitory effect against ROCK2 with IC50 lower than 500 nM. 22-24 showed a similar potency versus fasudil [88].

Recently, Lavogina et al.,[84, 89] have designed and synthesized a series of compounds that conjugates of 5-isoquinolinesulfonylamides and D-arginine-rich peptides. By introducing D-arginine into the N-(2-aminoethyl)-5-isoquinoline- sulfonamide, they successfully achieved several compounds with high selectivity for ROCK2 (i.e. the affinity of 27 towards ROCK2 is 160-fold higher than that towards PKA) [84, 89].

6. Future direction

Advanced as medical science is, it still remains a big challenge for human beings to overcome CNS disorders, for its complicated pathogenesis. Multiple signaling pathways are involved in the pathogenesis of CNS disorders, each contributing to the development of these disorders. Thus, it has become apparent that a "one-compound-one-target" neuroprotective drug may not be adequately powerful to modify the disorders [2]. One may assort to the use of cocktails of drugs or to develop bi- or multi-functional drugs to realize better modifying effects [90, 91]. Koumura et al.,[92] evaluated the combination therapy of fasudil and ozagrel, a thromboxane A2 (TXA2) synthase inhibitor, which is presently used in several countries for the treatment of acute cerebral infarction or the prevention of the cerebral vasospasms after SAH by antiplatelet and antithrombotic effects, in middle cerebral artery occlusion (MCAO) mice. Results showed that combination therapy of fasudil (i.p., 3 mg/kg) and ozagrel (i.p., 10 mg/kg) exhibits additive effects for neuroprotection after MCAO, implicating that the combination of fasudil and ozagrel may be a promising therapeutic strategy for stroke. Nevertheless, in the treatment of vasospasm after subarachnoid hemorrhage, combination of fasudil and ozagrel exhibits better efficacy than ozagrel alone but no better than fasudil [93, 94]. Furthermore, Chiba et al. [47], reported that bone marrow stromal cells transplantation and fasudil provide synergistic effects on axon regeneration after spinal cord injury. Other studies carried out to investigate the combination therapy of fasudil with other drugs have achieved some encouraging outcomes [95-98]. Combination strategy may become a fashion in the treatment with fasudil for CNS disorders. In addition, we also suppose that we could generate a small molecular by hybriding or fusing or chimera another pharmacophore with fasudil via aminoacylation to realize the high specificity towards ROCK2 and multi-targets. The new era of compounds may confer better brain penetration compared to fasudil, and enhance the therapy potency but minimize the toxicity of the drug(s). Anyway, further efforts should be exerted to confirm the hypothesis in coming days.

7. Conclusion

ROCK, discovered in 1996, has been proven to be involved in the pathogenesis of many CNS disorders such as SAH, AD and MS. In this review, we summarized the potential therapeutic effects of fasudil, the only clinically available ROCK inhibitor, in animal models and clinical applications of CNS disorders of SAH, AD and MS. Moreover, fasudil succeed in treating stroke and subarachnoid hemorrhage without causing any severe adverse effects. The dramatic effects of fasudil in animal models and/or clinical applications of CNS disorders make it a promising strategy to overcome CNS disorders in human beings. Fasudil, a non-specific ROCK inhibitor, also potently inhibits other protein kinases such as PKA. In order to get highly specific Rho-kinase inhibitors, Tamura M et al had synthesized several analogues of fasudil [86]. They found that substitution of the 2-position of hexahydro-1H-1, 4-diazepine and the 4-position of isoquinoline enhance the potency and specificity. Subsequently, they designed and synthesized a series of analogues of fasudil via amino acylation of the hexahydro-1H-1, 4-diazepine ring which lost inhibition against PKA but retained remarkable potency against ROCK on the basis of the complex structure of PKA and fasudil. In addition, specificity and potency of fasudil analogues against ROCK2 and other AGC kinases are summarized in Table 1.

Aberrant Rho/Rho kinase signaling has been strongly involved in the etiology of a wide range of CNS disorders, such as SAH, AD and MS. Therefore, inhibitors against Rho/ROCK signaling pathway are considered highly "drugable" and present as important targets for the treatment of these diseases. It is apparent that fasudil, with its diversely pharmacological activities and highly safety, will be applied in the treatments of diverse CNS disorders. Nevertheless, further studies will be warranted to demonstrate that fasudil and its analogues to be a new powerful weapon in the long war against CNS disorders.

8. Expert Opinion

CNS disorders have devastating effects on patients' quality of life. Till today, the therapies against these disorders are only able to attenuate the symptoms and fail in mitigating or preventing disease progression. Over the past decades, despite encouraging data from experimental animal models, almost all therapies have, to date, not been established in clinical routine. New approaches with disease-modifying activities are urgently needed. Rho/ ROCK is involved in a wide range of patho-physiological changes in the actin cytoskeleton and has been shown abnormal activation in a number of CNS disorders including SAH, AD and MS. This in turn renders inhibition of Rho/ROCK as a hopeful weapon against CNS disorders. Fasudil is the only clinically available ROCK inhibitor and has numerous beneficial effects including vascular dilation, neuroprotection, and promotion of axonal regeneration, which providing new insights into the treatment for CNS disorders. The dramatic effects of fasudil in animal models and/or clinical applications of CNS disorders make it a promising strategy to overcome CNS disorders in human beings. However, two problems should be solved before its clinical application for CNS disorders: one is the delivery and treatment duration, the other is the low selectivity, which inevitably cause some side effects.

The BBB penetration ability is the most key factor of effective drugs for CNS disease. Fasudil has low brain penetration ability, and some strategies, besides liposome preparation and myelin injection which were discussed above, can be used to overcome the shortage, such as making a pre-drug with higher BBB penetration in order to improve the brain biovialability of fasudil. In other hand, the duration of treatment for chronic CNS disorders usually is a long duration, which requires the drug should be low toxicity and few side effects. In clinic, fasudil is mainly used for SAH and the duration is one to two weeks. Obviously, more trails are necessary to test the possibility of using fasudil and hydroxyfasudil for other CNS disorders, such as AD and PD.

Fasudil has the equal blocking potency to ROCK1 and ROCK2, and also block other protein kinase at higher concentrations. As we discuss in the article, ROCK2 is mainly distributed in neuronal tissues, so it is necessary to develop more potent and selective inhibitors against ROCK 2 to exert better clinic usage with mild adverse effects. Maybe by hybriding or fusing or chimera with another pharmacophore with fasudil could generate "one-compound-multi-targets" fasudil derivatives with the high specificity towards ROCK2, multifunction and mild adverse effects, thus providing better clinical application.