Cyclosporine-A as a neuroprotective agent against stroke: Its translation from laboratory research to clinical application

Neuropeptides 45 (2011) 359–368

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Cyclosporine-A as a neuroprotective agent against stroke: Its translation from laboratory research to clinical application Mohamed M. Osman a, Dzenan Lulic a, Loren Glover a, Christine E. Stahl b, Tsz Lau a, Harry van Loveren a, Cesar V. Borlongan a,⇑ a Center of Excellence in Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612, USA b Department of Aerospace Medicine, MacDill Air Force Base, Tampa, FL 33621, USA

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Article history: Received 9 November 2010 Accepted 24 April 2011 Available online 17 May 2011 Keywords: Immunosuppressant Neuroprotection Cerebral ischemia Mitochondria Neurological disorders

a b s t r a c t Stoke remains a leading cause of death and disability with limited treatment options. Extensive research has been aimed at studying cell death events that accompany stroke and how to use these same cell death pathways as potential therapeutic targets for treating the disease. The mitochondrial permeability transition pore (MPTP) has been implicated as a major factor associated with stroke-induced neuronal cell death. MPTP activation and increased permeability has been shown to contribute to the events that lead to cell death. Cyclosporine A (CsA), a widely used immunosuppressant in transplantation and rheumatic medicine, has been recently shown to possess neuroprotective properties through its ability to block the MPTP, which in turn inhibits neuronal damage. This newfound CsA-mediated neuroprotection pathway prompted research on its use to prevent cell death in stroke and other neurological conditions. Preclinical studies are being conducted in hopes of establishing the safety and efficacy guidelines for CsA use in human trials as a potential neuroprotective agent against stroke. In this review, we provide an overview of the current laboratory and clinical status of CsA neuroprotection. Ó 2011 Elsevier Ltd. All rights reserved.

Contents 1.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Stroke as a leading cause of death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Etiology and pathophysiology of stroke. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Molecular mechanisms and inflammatory changes accompanying stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyclosporine A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Pharmacological properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Established uses of CsA in clinical practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. CsA neuroprotective properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CsA use in the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Preclinical studies on CsA in stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Laboratory studies on CsA in traumatic brain injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. CsA indications for other neurological disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Human trials: safety and efficacy outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Adverse effects of CsA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements and disclosure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding author. Address: Center of Excellence in Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, MDC 78, Tampa, FL 33612, USA. Tel.: +1 813 974 3988; fax: +1 813 974 3078. E-mail address: [email protected] (C.V. Borlongan). 0143-4179/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2011.04.002

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1. Introduction

1.2. Etiology and pathophysiology of stroke

1.1. Stroke as a leading cause of death

Cerebrovascular stroke occurs when the blood supply to the neural tissue of the central nervous system (CNS) is interrupted. The rising incidence is likely due to increase in the risk factors that predispose to compromised integrity of cerebral blood supply; hypertension, diabetes and smoking are the most commonly seen modifiable risk factors, others include atrial fibrillation, transient ischemic attacks (TIAs), hyperlipidemia, and ischemic heart disease. Genetic predisposition is also implicated as a significant risk factor in 40% of cases. There are two types of stroke; ischemic and hemorrhagic (Thrift et al., 2001), and since the management of both conditions is very different, distinction between the two subtypes is critical. Use of modern investigative modalities, such as computerized tomography (CT and magnetic resonance imaging (MRI), have greatly enhanced the rate, accuracy and speed of diagnosis. Hemorrhagic strokes are usually secondary to the rupture of small hypertensive micro-aneurysms called Charcot-Bouchard aneurysms, or to angiopathy such as amyloid angiopathy. Occasionally, patients suffering from hemorrhagic stroke may require surgical intervention for hematoma evacuation. In contrast, the common causes of ischemic stroke include cardiac emboli, carotid atherosclerotic disease, and small vessel disease. About 80% of stroke cases are of the ischemic variant. Arterial occlusion leads to decrease regional blood supply. As regional blood supply drops below 18 cc/100 g/min, electrical conduction of neurons fails. When blood flow drops below 10 cc/100 g/min, irreversible neuronal damage occurs (Astrup et al., 1981). The physiology of the neuron forms the basis of acute stroke intervention. In acute stroke, irreversible damage often occurs in the center of ischemia, the function of surrounding brain tissue is only temporarily impaired. This ischemic tissue is also known as the ‘‘ischemic penumbra’’; in the absence of intervention to restore blood flow, this ischemic area will proceed to irreversible damage or death. However, when therapy is initiated in a timely manner to restore blood flow, the ischemic penumbra is potentially salvageable tissue (Donnan et al., 2008; Jauch, 2010).

Stroke is second only to ischemic heart disease as the most common cause of death, annually claiming 9% of deaths worldwide. The World Health Organization estimates 15 million cases of stroke each year, five million of which are fatal, and five million more suffer permanent disability. In the United States, stroke is considered the third leading cause of death, with over 795,000 reported cases each year (Marsh and Keyrouz, 2010). To this day, treatment modalities for stroke are limited and mainly targeted at preventing complications and reduction of risk factors. Apart from the use of fibrinolytic agents (tissue Plasminogen Activator or tPA), there is no medical therapy that restores neural function and protects from ischemic damage. Use of these treatments is also limited by the narrow therapeutic window associated with stroke, during which there is hope of salvaging neurological integrity (Donnan et al., 2008). According to the acute stroke treatment guidelines published by the American Heart Association (Adams et al., 2007), the mainstay of acute stroke treatment focus on revascularization, either with agents such as tPA or with endovascular mechanical device. To date, the regular use of neuroprotective agents has not been established due to a lack of clinical data. In general, intravenous tPA must be given within 3-h after symptoms onset and 6-h for intra-arterial administration. This excludes its use in many patients that present to the hospital. Furthermore, numerous contraindications to the use of fibrinolytic agents that may increase the risk for intracerebral hemorrhage (Marler, 1995) excluded many stroke patients from receiving tPA. Further expansion of treatment armamentarium is necessary to improve morbidity and mortality in this group of devastate and desperate patients. The MPTP is thought to play a critical role in ischemic neuronal cell death (Schinzel et al., 2005). Cyclosporine A (CsA), which has been used as an immunosuppressive agent in transplant medicine, has been proposed to block the MPTP and protect the rat brain from stroke (Leger et al., 2010; Borlongan et al., 2002a). Emerging evidence suggests CsA might be a promising agent in treating stroke and further investigation in the clinical setting should be performed. The potential clinical applications of CsA in stroke are enormous, not only it maybe useful in patients that are not candidates for tPA, but its neuroprotective mechanism may also be complementary to tPA treated stroke patients as well. However, the possible adverse effects of CsA should be recognized as well, and the eventual clinical role in treating stroke will be based on the risk and benefit ratio.

1.3. Molecular mechanisms and inflammatory changes accompanying stroke In order to properly develop a treatment that provides neuroprotection and prevents cell death after stroke, it is vital to understand the mechanism of stroke-induced cell death on a chemical and molecular level. The mitochondria are centrally involved in the development of stroke and cell death. Deficits in the production

Fig. 1. Chemical structure of Cyclosporine A. CsA is an active metabolite found to be a cyclic undecapeptide, rich in hydrophobic amino acids, neutral, insoluble in n-hexane and water, but very soluble in all other organic solvents. The structure and conformation of CsA was determined by chemical degradation using X-ray crystallographic analysis and by two-dimensional nuclear magnetic resonance imaging.

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of ATP (adenosine tri-phosphate) seen in ischemic tissue are the main reasons for the cell death; this is due to the impaired metabolic function of the mitochondria. Many studies carried out on rats and mice with middle cerebral artery occlusion (MCAo) stroke models have greatly implicated the MPTP in triggering apoptosis and cell death. Leakage of solutes through the permeability transition pore is thought to be due to high levels of calcium, which are caused by oxidative stress and depletion of adenine nucleotides (Leung and Halestrap, 2008). (Fig. 1) Despite extensive research, the composition of the MPTP remains poorly understood. It is, however, suggested that the voltage-dependent anion carrier (VDAC) in the outer membrane, cyclophilin D (CypD) in the matrix and, to a lesser extent, the adenine nucleotide translocase (ANT) in the inner mitochondrial membrane, are highly involved in the process (Sims and Muyderman, 2010). Studies on mice that lack the expression of CypD have confirmed its integral role since these animals failed to show increased MPTP and developed much smaller infarcts than their CypD expressing counterparts (Schinzel et al., 2005). They did however develop MPTP at very high calcium levels. Leung and Halestrap (2008) confirmed in their studies the role of CypD in the development of MPTP, proposing that calcium triggered activation of CypD and subsequent interaction with the mitochondrial phosphate carrier (PiC) and ANT leads to the pores activation. The exact mechanism is still unclear and further investigation is being conducted.

2. Cyclosporine A 2.1. Pharmacological properties Cyclosporine A (CsA) is an undecapeptide isolated from the fungus Tolypocladium inflatum Gams, which is grown from a soil specimen from Norway (Fig. 2). Its immunosuppressive properties were initially discovered by Borel and his team (1976), who demonstrated that CsA was able to suppress the activity of T-lymphocytes (Borel et al., 1976). CsA exhibits unpredictable pharmacokinetics. It can be administered orally or intravenously, and is incompletely absorbed by the small intestines. The level of bile secretion and intestinal motility affect the absorption of CsA. Bioavailability after oral administration varies from 20% to 50%, with a peak concentration 3–4 h after administration. Once in the circulation, it is highly bound to erythrocytes and to a lesser extent to leukocytes. CsA is almost exclusively metabolized by the liver and is excreted through the bile (Khan, 2008). Since this discovery, the clinical use of CsA was initially reserved as an immunosuppressant in transplant medicine due to its ability to alter T-lymphocyte activity. It has also been used to reduce the incidence of transplant rejection by the host. In the cell, it binds to its cytosolic receptor, Cyclophilin A (CypA). The complex of CsA–CypA then binds to the calmodulin-stimulated protein phosphatase calcineurin, inhibiting calcineurins serine–threonine phosphatase activity. By preventing calcineurin-mediated dephosphorylation, CsA inhibits the translocation of the nuclear factor of activated T cells (NFAT) family of transcription factors from the cytoplasm to the nucleus of activated T cells. The NFAT group is involved in the transcriptional activation of the genes encoding interleukin (IL)-2, IL-4 and CD40L; thus, inhibition of the NFAT by CsA results in a specific inhibition of interleukin production in the T cell (Nomura et al., 1994). Several other cyclophilins have been discovered, such as cyclophilin B, C, and D. CypD is located in the mitochondria and, unlike A, B, and C, it is involved in neuroprotection (Snyder et al., 1998). Cyclophilins belong to a group of molecules also known as immunophilins that bind immunosuppressant agents such as CsA. Since

Fig. 2. Factors regulating mitochondrial permeability transition (MPT) pore opening. Oxidative stress, low pH, or Ca2+ overload in the mitochondrion during injury leads to the increased MPT. Binding of CsA to CypD prevents the pore opening. Proposed mechanism is that CsA binds to CypD and inhibits-induced oxidative stress leading to protection.

many of the immunophilins have neurotrophic and neuroprotective effects, they are also referred to as neuroimmunophilins. Neuroimmunophilins not only inhibit calcineurin, but they also block MPTP and mitochondrial mega-pore formation. 2.2. Established uses of CsA in clinical practice In light of its T-cell immunosuppressive properties, CsA is currently established in clinical practice for a number of T-lymphocyte mediated conditions, mainly in the venue of transplantation. CsA is used for the prevention of rejection after organ transplantation, primarily in cases of liver, heart, and kidney transplants. Moreover, the drug is also indicated for management of multiple rheumatic, dermatologic, gastrointestinal, and ophthalmologic conditions. CsA revolutionized transplant medicine upon introduction and for more than two decades its use has been established in kidney, heart, liver, and bone marrow transplants (White and Calne, 1982). In addition to transplantation, CsA is also used in the treatment of many dermatological conditions such as severe psoriasis, pyoderma gangrenosum, vasculitis, scleroderma, and eczemas (Berth-Jones, 2005; Callen, 2001). CsA has also been used in cases of steroid resistant ulcerative colitis in multiple trials, and has shown to be effective in approximately 40% of patients when used alone or in combination with corticosteroids (Hanauer, 2004). With regard to ophthalmological practice, Baiza-Duran and colleagues concluded after studying 183 patients that CsA use reduced signs and symptoms in patients with moderate to severe dry eye disease (Baiza-Duran et al., 2010). On the other hand, significant numbers of adverse effects limit its usage (Ramos-Casals et al., 2010). Gerloni and his team had shown that CsA use also displayed some benefit in treatment of juvenile chronic arthritis (JCA); however, his subjects did show frequent but mild and reversible adverse effects to the treatment (Gerloni et al., 2001).

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Given the successful clinical applications of CsA, limited adverse effects, and encouraging data in animal stroke models, this agent should at least be considered for the treatment of stroke in the clinical setting. As mentioned in the introduction, there are limited treatments available for acute stroke and many patients are not candidates for existing medical treatment such as tPA due to a narrow time window and various contraindications. Although accumulating experimental and clinical research evidence has demonstrated neuroprotective effects in acute ischemic stroke, additional safety and efficacy data are necessary to test these neuroprotective agents alone or in combination with measures to restore perfusion (i.e., tPA) before solid clinical guidance can be established that such neuroprotective therapies are effective in improving outcomes after stroke. Accordingly, the American Heart Association does not recommend the use of neuroprotective agents (Adams et al., 2007). Furthermore, the NINDS tPA study (Marler, 1995), which is a randomized and double blind clinical trial, revealed that despite the use of tPA, patients with large vessel occlusive disease and cardioembolic stroke with initial median NIH stroke scale score of 14, only <50% has favorable outcome in 3 months and 9–12% of patients that received tPA suffered posttreatment intracerebral hemorrhage. In summary, CsA should be considered for further investigation as a neuroprotective agent for acute stroke patients for 4 reasons: (1) limited treatment options are currently available; (2) many patients are not candidate for treatment for stroke such as the use of tPA; (3) despite current treatment, the vast majority of stroke patients still have an unfavorable long term outcome; (4) CsA acts differently than existing treatment regimen. 2.3. CsA neuroprotective properties Although the protective effects of CsA were first discovered in T-lymphocytes, the brain has about a 20-fold greater tropism for CsA compared to the T-cells (Goldner and Patrick, 1996). The benefits of oral CsA as a neuroprotective agent are achieved only with a high dose (i.e., >10 mg/kg) and chronic administration of the drug. However, such high dosage and chronic injection of CsA produces negative side effects such as nephrotoxicity and hepatotoxicity, among others. Thus, a major limiting factor in producing the therapeutic effects of CsA is its ability to cross the blood brain barrier (BBB). It has been demonstrated that CsA protective effects may be enhanced by mechanical disruption of the brain parenchyma (Borlongan et al., 1998; Hayashi et al., 2009; Leventhal et al., 2000). One novel approach involves the administration of endogenous ligands or their analogs to increase permeability of the BBB by activation of receptors on the endothelial cells comprising the BBB (Inamura and Black, 1994; Nomura et al., 1994). Selective B2 bradykinin receptor agonist, Cereport, has been shown to transiently increase the permeability of the BBB (Bartus et al., 1996). The P-glycoprotein (P-gp) is a membrane transporter encoded by the MDR1 gene that contributes to the blood brain barrier by transporting out various drug molecules, including CsA, from the cells. This molecular mechanism contributes to drug resistance of tumors to many chemotherapy agents. Double knockout mice mdr1a ( / ) injected with radioactive isotopes showed a 20– 50-fold increase of various drugs including digoxin and CsA in the brain (Schinkel et al., 1995, 1996). However, the increase level of drugs in the brain may also contribute to neurotoxicity. Compared to the wild type, the mdr1a ( / ) mice have a 100-fold increased sensitivity to the ivermectin and 3-fold increase to vinblastine. This observation explains some of the adverse effects seen in patients treated with both chemotherapy agents and P-glycoprotein inhibitors (Schinkel et al., 1994). Future research should further elicit how the MDR1 gene polymorphism will affect the efficacy of CsA on the ischemic brain.

Another target of calcineurin that is blocked by CsA is nitric oxide synthase (NOS). Activated calcineurin dephosphorylates NOS, which is responsible for the production of nitric oxide (NO). Nitric oxide is an intracellular messenger when synthesized in moderate amounts, however excessive levels can be toxic. Nitric oxide is a well-known neurotoxin induced in the case of glutamate neurotoxicity and is a free radical that can inactivate proteins and enzymes (Dawson et al., 1996). It can also interact with O2 and create peroxynitrite (ONOO ), a highly neurotoxic molecule (Wang et al., 2010). CsA inhibits a crucial step in MPTP formation, which, subsequently, will prevent the activation of apoptotic cascade events in stroke. In support of this model is the current evidence that CypD has a major role in ‘‘energy failure’’, but not in glutamate excitotoxicity; therefore, CypD is tied to the mitochondrial dysfunction rather than increased intracellular calcium levels (Malouitre et al., 2010; Schinzel et al., 2005). According to multiple studies, CypD-deficient mice displayed a reduction in brain infarct size after acute middle cerebral artery occlusion and reperfusion (Schinzel et al., 2005; Wang et al., 2009, 2010). This finding is reinforcing the evidence that CsA inhibits activation of MPTP and pore formation (Nicolli et al., 1996) by inhibiting matrix CypD interaction with pore proteins (He and Lemasters, 2002). CsA has been demonstrated to produce neuroprotective effects in both in vivo and in vitro models of excitotoxicity (Santos and Schauwecker, 2003; Snyder et al., 1998). Protective effects of CsA in the case of glutamate excitotoxicity are evident on multiple levels. As previously mentioned, CsA prevents increased MPTP and preserves normal mitochondrial function. However, in the case of glutamate excitotoxicity it also acts through inhibition of calcineurin-mediated release of synaptic vesicles (Kaminska et al., 2004). By inhibiting calcineurin, CsA prevents phosphorylation and subsequent activation of dynamin I and synapsin I, both of which have been implicated in the regulation of neurotransmitter release (Cousin and Robinson, 2001; Kaminska et al., 2004). Additionally, it also blocks calcineurin-mediated dephosphorylation of NOS subsequently blocking NOS activation. Activated NOS is responsible for the production of nitric oxide, which plays an important role in neurotransmitter release from synaptic vesicles (Dawson et al., 1993, 1996; Kaminska et al., 2004). An equally critical cell death pathway that is blocked by CsA entails the apoptosis signaling mechanism. This mechanism involves Ca2+-induced activation of Bcl-2-associated death promoter (BAD). BAD is a Ca2+-calcineurin sensitive pro-apoptotic Bcl-2 protein, which upon activation, translocates from the cytosol to the mitochondria to initiate cytochrome c release and the apoptotic cascade (Shou et al., 2004; Wang et al., 1999). Calcineurin inhibitors like CsA block the apoptotic response linking calcineurin activation and the subsequent translocation of BAD to cell death (Wang et al., 1999). The full extent of action and neuroprotective effects of CsA are still being clarified. There is emerging evidence that CsA could induce neuroprotective effects through other pathways and stimulate the production of neurotrophic factors (Costantini et al., 1998; Gabryel and Bernacki, 2009). After forebrain ischemia, an increase of brain-derived neurotrophic factor (BDNF) was observed in animals treated with CsA compared to animals that did not receive treatment (Miyata et al., 2001). Additionally, our group showed that CsA-treated, 6-hydroxydopamine (6-OHDA) lesioned Parkinsonian animals displayed significantly higher general spontaneous locomotor activity than control animals at after about a week of daily CsA intraperitoneal (IP) injection (10 mg/kg), which coincided with a significant increase in TH-immunoreactive neurons in the nigra and suppressed calcium-phosphatase calcineurin activity, indicating robust neurotrophic activity (Hui et al., 2010) and an inhibition of host immune response, respectively

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(Borlongan et al., 1999). CsA’s neurotrophic factor activity attenuated the dopaminergic depletion produced by the neurotoxin 6-OHDA could also be enhanced by transient permeabilization of the blood brain barrier, allowing lower doses of CsA (1 mg/kg) to similarly exert neuroprotective effects against experimental model of Parkinson’s disease (Borlongan et al., 2002b; Borlongan and Emerich, 2003). We also extended similar neurotrophic factor effects of CsA in the cholinergic system, in that adult rats treated with CsA (10 mg/kg per day, i.p. for 9 days) also exhibited significantly reduced septal CN expression in combination with enhanced levels of septal choline acetyltransferase immunoreactivity compared to the controls (Borlongan et al., 2000). Other models of neuronal injury, such as stroke, spinal cord injury, and traumatic brain injury, also implicate the neuroprotective role of CsA via facilitation of neurotrophic factor activity (Sheehan et al., 2006; Hayashi et al., 2005; Kaminska et al., 2004; Gold, 2000).

3. CsA use in the CNS 3.1. Preclinical studies on CsA in stroke In light of its neuroprotective properties and prevention of cell death, CsA was seen as having potential value in the management of stroke. One of the earliest studies was carried out by Paterson and colleagues who tested its effects after subarachnoid hemorrhage in dogs (Peterson et al., 1990). Their aim was to test the effect of the drug on vascular spasm following subarachnoid hemorrhage. The study was carried out by administering prophylactic CsA in the subjects and measurement of basilar artery diameters after hemorrhage. Studies investigating the neuroprotection in stroke models were not far behind. Early promising findings were those of Wakita colleagues (1995). They studied the effect of IP injections of CsA on rats following bilateral common carotid artery ligation. Their study used 40 animals, 22 of which received daily 10 mg/kg CsA IP injections starting one day pre-operation, continuing after ligation for 14 days and every third day until completion of the 30 day period. The remaining 18 were used as a vehicle control group. An additional group of 5 animals were added as a sham control group. Furthermore, to investigate dose related effect, an additional group of 8 animals received 5 mg/kg CsA, and 9 received 15 mg/kg, these animals were killed 14 days after ligation. The subjects were harvested on days 7, 14, and 30 and the brain sections were studied for white changes using a 4-grade scale. Their results showed much lower CD4 and CD8 lymphocyte infiltration and reduced microglial and macrophage activation in CsA treated animals compared to the vehicle control group. There was no significant difference between the 10 mg/kg and the 15 mg/kg; groups, but the 5 mg/kg group showed reduced protective effects. A more focused study showed the direct effect of CsA on brain ischemia demonstrated in the rat stroke models. Yoshimoto and his team 1999 used the middle cerebral artery 2-h occlusion (MCAo) on the rats and compared the effect of intravenous injection of 10 mg/kg CsA after reperfusion, while using one of three methods for disruption of BBB: the injection was either (1) accompanied by ipsilateral needle lesion, (2) by raising the IV dose to 50 mg/kg in order to overload the BBB CsA transport mechanism, or by (3) intra-arterial injection of 10 mg/kg directly into the carotid artery with or without prior mannitol, the different regimens were also administered at different intervals at 0 min, 5 min, 1 h, 3 h, and 6 h delay. The results showed reduction in infarct sizes with variations according to the regimen used; IV injection alone showed no reduction to infarct size; however, the needle lesion group showed infarct volume 40% that of the control. Increasing the dose to 50 mg/kg successfully reduced infarcts to 20% of

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control but these animals showed higher levels of toxicity. The intracarotid injection group showed the best results with reduced size to 10% that of control. Better outcomes were also seen in groups injected at 5 min with efficacy lowering in the longer interval groups at 1, 3, and 6 h, respectively. These studies with CsA were compared to Tacrolimus (FK506), another immunosuppressant known to enhance phosphorylation of nitric oxide synthase and protect against glutamate neurotoxicity (Dawson et al., 1993). The difference between CsA and FK506 is that CsA does fully suppress brain damage in rat forebrain ischemia when it is allowed to pass the BBB, however, FK506 is considered less effective. In addition, CsA shows more potent anti-ischemia effects than FK506 (Uchino et al., 2002). Comparison to the FK506 groups showed only minimal difference in favor of CsA, but this was not considered by the authors to be a conclusive finding. This study highlights a very important point that directly addresses the obstacle of CsA BBB permeability. Disruption of the BBB showed a substantial increase in the efficacy of neuroprotection achieved by CsA. Moreover, there is a significant difference seen according to the method of disruption. Many methods have been studied in order to achieve effective and safe disruption of the BBB. Bradykinin receptor agonists have shown promising results and provide a minimally invasive technique that is realistic and have been safely applied in clinical practice for enhancing delivery of pharmacologic agents across the BBB (Borlongan et al., 2002b; Borlongan and Emerich, 2003). A further study based on gerbil ischemia models showed that CsA, when compared to FK506, had a greater neuroprotective effect on the insulted brains (Domañska-Janik et al., 2004). In this study, the protective effect was demonstrated by the inhibition of cytochrome C release in the cells after ischemia. The tested area was the CA1 region of the hippocampus, where CsA treatment showed marked reduction in neuron loss. The effect is, however, limited by the time of administration. Animals that were injected IC immediately after ischemia were effectively infarct free. When the injection is delayed up to 6 or 24 h, the protective effect completely disappeared; moreover, when the does is lowered from 5 mg/kg to 2.5 mg/kg there is no positive response seen to the treatment. When comparing CsA to FK506, the investigators found that collectively almost 75% of neurons were saved from ischemic injury after CsA treatment, and not after FK506 treatment. In light of these findings, CsA could prove to be more effective as a neuroprotective drug in cases of ischemia when compared to FK506. Both drugs inhibit calcineurin activity after ischemic insult, but a study has demonstrated that CsA completely suppressed mitochondrial swelling and cytochrome c release while FK506 only exhibited partial inhibition (Uchino et al., 2002). The exact reason behind this is yet to be completely explained. It has been demonstrated that while an intact BBB greatly hinders penetration and effectiveness of CsA treatment, that is not the case with FK506 (Uchino et al., 2002). The therapeutic window for CsA neuroprotection may vary depending on indication (TBI, SCI). Some laboratory studies report (Sullivan et al., 2011; Sullivan et al., Exp Neurol, 2000 and Neuroscience, 2000; Büki et al., 1999; Yoshimoto and Siesjö, 1999) therapeutic effects ranging between 3–6 h and even at 8 h after injury, indicating direct clinical use. Although this may complicate clinical research protocols due to consent issues, for clinical care this would be a great improvement compared to current tPA time limits. Another study raises a very troubling finding in relation to the dose dependent actions of CsA. This study investigated the effects of CsA injection IP on 3 groups of knockout mdr1a mice. In the mdr1a knockout mice model, the blood–brain barrier is partially impaired. The mdr1a gene normally produces the major p-glycoprotein in the blood–brain barrier, but without this gene the

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knockout mice show increased drug levels, mainly in the brain (Murozono et al., 2004). Using the knockout mdr1a model seems to be a good model because it directly affects the blood brain barrier, which has to do with one of CsAs limitations. The drug was given to 2 groups in the doses of 1 mg/kg and 10 mg/kg after 30-min ischemia following the established MCAo model, the third group was given only vehicle as a control group. After 48 h the low dose group exhibited reduced infarct size compared to the vehicle treated group with a mean infarct volume of 16% and 26%, respectively. The alarming find was that the 10 mg/kg group developed a larger infarct size with a mean volume of 40%. This find uncovers the fact that, not only is CsA a neuroprotective drug, it can also exhibit neurotoxic effects in large doses. Further investigation is needed to establish solid parameters on that aspect (Murozono et al., 2004). Similar toxic effects have been observed in human patients receiving CsA after transplantation (up to 60%). The mechanism of these toxic effects remains unclear, although suggested theories based on magnetic resonance spectroscopy (MRS) claim that the effects are due to mitochondrial inhibition with subsequent drop in the ATP production and lactate accumulation with over-production of ROS products. These effects seem to be directly related to the dosage of the drug and the oxygen levels in the neural cells (Serkova et al., 2004). These findings are alarming and raise major concerns regarding possible toxicity fears; more research is required in order to establish the safe regiment for usage as a neuroprotective a role. When seen in human transplant patients, it raises the question of whether such effects are due to the prolonged use of the drug for extended treatment regimens, or if these are also seen in a more short-term setting. The rising concern regarding the high toxicity associated with CsA dosage effective in providing neuroprotection has directed the search toward finding an alternate regimen that would reduce these toxic effects. The first study to report the success achieved neuroprotection with low dose CsA administered with methyl prednisone (MP). These effects, however, were partial and transient (Yu et al., 2004). The study was carried out on 30 rats using the MCAo protocol, randomly assigned after 1-h occlusion into groups according to the therapeutic regimen. Treatments given were low dose CsA alone, MP alone, low dose CsA and MP, high dose CsA, and vehicle alone. Treatments were initiated 3-h after reperfusion, followed by subsequent injections at 24, 48, and 72 h, all injections were given IP. Behavioral testing was performed daily and histological testing was carried out on the 3rd day. The team found significant reduction in stroke size and motor deficits in the high dose group, the low dose CsA and MP group showed reduced motor deficit only on the first day, and did not show any reduction in stroke size upon histology 3 days after surgery when compared to CsA alone and vehicle groups. They concluded that low dose CsA when given with MP following 1-h ischemia, can produce neuroprotection, although these effects are only partial and transient. This conclusion opens the door to the possibility of modifying the treatment regimens and use of adjuvant drugs in order to use lower doses of CsA and reduce toxic effects. Further study is needed for establishing the proper dosage and administration time to maximize efficacy while avoiding the toxicity associated with the high dose regiments (Borlongan et al., 2005). The idea that isolating the action of CsA on CypD and preventing calcineurin activation can reduce the toxic effect of CsA has been gaining some popularity since both molecules have different binding sites, and the development of CsA analogs like NIM811 (Melle4-cyclosporin) and UNIL025 (MeAla3EtVal4-cyclosporin) that perform this function are under investigation (Hansson et al., 2004). The CsA analog FR901459 was studied in hopes of reducing the side effects seen with CsA therapy. This compound has been shown to reduce cell death, but the mechanism of its neuroprotective properties are not yet understood (Muramatsu et al.,

2007). The study was conducted in a two-arm fashion, testing the effect of FR901459 versus CsA on global and focal ischemia. For the global ischemia gerbils were used and the drugs were given intravenously after transient forebrain ischemia surgery. The stroke model used was through bilateral occlusion of common carotid arteries; histological assessment of harvested brains was done 4 days after reperfusion, sham operated animals were also used in this study as controls. The focal ischemia group employed rats that were operated on using the MCAo model and drug administration was done intravenously in one group and intracerebroventricular in the second group. Histological assessment was done 24 h after surgery. Both study arms were also assessed for immunosuppression via mixed lymphocyte reaction assay and for mitochondrial swelling and protein concentration. The results showed that FR901459 was 7 times less potent in producing immunosuppression than CsA, regarding the neuroprotection aspect, however FR901459 was also 7 times less potent than CsA in providing neuroprotection. These results were seen in both arms of the experiment and did not differ when the drug was directly injected intracranially rather than intravenously. The conclusion here is that although the lower toxicity seen with FR901459 is promising, it is also held back by its reduced efficacy. The compound’s ability to suppress ischemic cell damage was mainly due to its inhibition of mitochondrial swelling, which when compared to CsA, could raise the question of whether the role of calcineurin inhibition in neuroprotection is more important than previously believed. This study not only raises an important issue, but also questions whether the neuroprotective actions of CsA could in fact be isolated from its toxic immunosuppressive effects. The use of two different animal species could be regarded as unorthodox, but no significant discrepancy is seen in the results for both groups. More work is needed to answer the question of safety with CsA use. 3.2. Laboratory studies on CsA in traumatic brain injury Current studies based on animal data provide compelling evidence of therapeutic benefits of CsA in TBI, but the outcome indices are heterogeneous with respect to the animal model of TBI as well as route, dose, and timing of CsA administration. Similarly, clinical studies (Phase II trials) adapting almost identical patient inclusion criteria have demonstrated safety of CsA use in TBI, but the clinical trials are also heterogeneous based on study design especially in regards to the variable timing of CsA administration after TBI. Most of them showed the benefits of CsA use in TBI as in animal models. Equally important to note is that most of these studies explored the efficacy of CsA in models of severe traumatic injury. However, many patients suffer moderate and mild TBI, indicating the need to comparatively explore CsA effects in animal models that correlate to mild, moderate, and severe TBI in humans. Moreover, CsA success in TBI models has prompted new research and testing on analogs of CsA, such as NIM811, that have decreased nephrotoxic side effects (Rosenwirth et al., 1994). It is important to note that despite the absence of full characterization of CsA, efficacy, and the mechanism of action, the overall preclinical data strongly supported initiation of small clinical trials. One of the first studies on human subjects explored pharmacokinetics and CsA disposition in TBI. All of the clinical Phase II trials confirmed the safety of CsA for use in TBI patients. As in animal studies, clinical trials are heterogeneous in respect to the study design, timing, and length of administration of CsA after TBI (Empey et al., 2006). 3.3. CsA indications for other neurological disorders Promising prospects have suggested the efficacy of CsA as a neuroprotection agent in prevention and control of other CNS diseases. The success of CsA in cases of TBI prompted the investigation

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of its use in traumatic spinal cord injury. Inflammatory processes associated with free radical release, glutamate excitotoxicity, and lipid peroxidation seen in these cases contributes significantly to the neuronal damage and clinical outcome. The beneficial effects of CsA were studied in rat models with severe spinal cord injury using specialized dosage schemes to guard against feared toxicity (Ibarra et al., 1996). Results showed that a dose of 2.5 mg/kg every 12 h produced 66% decrease in lipid peroxidation, the best results were seen when the drug was administered in the first 6 h after injury (Diaz-Ruiz et al., 1999). The potential therapeutic use of CsA in Parkinson’s disease was also examined, with CsA shown to improve locomotive function in parkinsonian rats and may even have a more beneficial effect when used alongside neural transplantation (Borlongan et al., 2002b). Further investigation on induced dyskinesia in rat models has demonstrated that CsA both restores and protects dopaminergic neurons (Iida et al., 1998). Similar effects were also seen in cases of Huntington’s disease (HD) where laboratory data showed that CsA treatment protects medium sized spiny neurons from 3-nitropropionic acid, a toxin that produces mitochondrial damage similar to that seen in cases of HD (Ouary et al., 2000). CsA was also found to have protective effects in Alzheimer’s disease (AD); cell culture-based and in vivo studies have shown that MPTP suppression can prevent or slow down the neurodegenerative process seen in AD (Mattson, 2003). Based on its key immunosuppressive property, CsA was also examined in multiple sclerosis (MS). Results showed some improvement in patient’s baseline neurological functions and reduction in the progression rate of the disease. The immunosuppressive dose of the drug failed to cross the blood brain barrier while higher doses showed increased toxicity (Group, 1990). The studies were unfortunately crippled by frequent drop outs due to high toxicity rates. Newer regimens with better tolerability and better toxicity levels may once again reopen the door for reevaluating the use of CsA in MS (Oger, 2007). Despite the poor results seen in cases of MS, other CNS autoimmune disorders show good response to CsA treatment, including myasthenia gravis (Tindall et al., 1987). Overall, the use of CsA in these conditions appears promising, but significantly limited by the poor penetration of CsA to the BBB and high toxicity seen with increased dosage regimens. 3.4. Human trials: safety and efficacy outcomes CsA has been used for more than two decades now in humans and is part of many established regimens as an immunosuppressant in post-transplant patients (Cholongitas et al., 2010; Hesselink et al., 2004; Kahan, 1993) and other autoimmune disorders (Berth-Jones, 2005; Gerloni et al., 2001; Hanauer, 2004), as discussed earlier. The new direction here is the use of the drug for its neuroprotective properties for stroke patients. This model of therapy has not yet been applied and guidelines for its application as still being developed. The closest therapy model that is present is the currently undergoing experimentation for the use of CsA as a neuroprotectant in TBI. Most of the neuroprotection focused human studies on CsA were on efficacy and safety as part of Phase II trials. Multicenter Phase III trials are still in progress, but there has been some promising data coming from Phase II trials already. All of the four Phase II trials showed safety of CsA for neuroprotection after TBI. This is comforting since the mechanism behind the use of CsA in stroke and TBI is basically the same. One of the first studies on human subjects, by Empey et al., explored pharmacokinetics and CsA disposition in TBI (Empey et al., 2006). This was a Phase II, randomized, dose-escalation, placebo-controlled trial with 30 patients. The patients were studied using a 3-dose regimen and a controlled placebo; the groups received either 0.625 mg, 1.25 mg, 2.5 mg/kg/dose of CsA or placebo intravenously every 12 h for 72 h. Their data showed

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that pharmacokinetics in the TBI population is different from populations in previous studies, which focused on healthy individuals or the transplant patient population. The TBI population had whole blood clearance, steady state volume of distribution, and half-life higher than in other published reports (Empey et al., 2006). Mazzeo and colleagues showed that TBI is followed by a significant decrease in cellular immune response and T-cell activation (Boddie et al., 2003; Mazzeo et al., 2006). Being immunosuppressant itself by blocking T-cell activation and proliferation, CsA could present a serious problem in already immunosuppressed patients with stroke who are likely to have preexisting chronic illness. Mazzeo and co-workers (2006) conducted a randomized, placebo-controlled study to evaluate the safety and tolerability of CsA and its effect on cell-mediated immunity in 59 severe head injury patients. Thirty-six patients received an infusion of 5 mg/kg of CsA over 24 h within 12 h after injury. Another eight patients from this cohort received an additional 5 mg/kg 24-h infusion of CsA with 200 mg of ketoconazole via a feeding tube to enhance the bioavailability of CsA. Twelve patients received a placebo within 12 h after injury. Although greater than 60% of all patients had reduced baseline lymphocyte count, this was considered to be associated with the increase in pulmonary infections rather than treatment with CsA. Baseline, 48-h, and 72-h lymphocyte count, CD 3, CD4+ and CD8+ count as well as CD4/CD8 ration did not differ between the CsA-treated group and placebo. They also documented T-cell infiltration in brain parenchyma in four patients undergoing craniotomy for decompression (Mazzeo et al., 2006). This study concluded that CsA administration was not associated with a decrease in T-cell immunity. Instead, compromised immunity was related to severe head injury itself, as previously documented in other studies. It still is unclear why a potent immune suppressant like CsA had a paradoxical effect on the cellular immunity and did not further lower immune competence in patients with traumatic brain injury. Further investigation is still necessary with a larger sample size for more definitive conclusions. Another drawback of this study is that it was not blinded and the placebo group had a significantly better Glasgow coma score (GCS) than the CsA cohort, which suggests a possible selection bias. Hopefully, Phase III trials will offer some more insight on the nature of CsA interaction with cell-mediated immunity in these neurologically compromised patients. An additional study that directly evaluated the safety and efficacy of CsA was performed by Hatton et al. This study was a Phase II, randomized, double-blind, dose-escalation trial with 40 patients enrolled (Hatton et al., 2008). This was a well conducted Phase II trial which showed that CsA administration was safe in patients with TBI, including better outcomes seen with a continuous infusion of 5 mg/kg of CsA with an initial loading of 2.5 mg/kg of CsA. Such information should be very useful for Phase III trials in progress, since it offered the most effective administration dose associated with better outcomes. Hopefully such dosing might yield statistical significance because of the much larger sample size used in Phase III trials. The most recent dual-center study from National Institutes of Health – National Institute of Neurological Disorders and Stroke (NIH-NINDS) and Virginia Commonwealth University assessed safety and tolerability of single infusion 5 mg/kg of CsA within first 12 h after injury (Mazzeo et al., 2009). Infusion of CsA was administered over 24 h. This double blind, placebo-controlled trial had initially enrolled 50 patients. This study demonstrated that CsA is safe for use within first 12 h of injury as the single infusion of 5 mg/kg. However, the authors found transient rise in BUN levels 24 and 48 h and transient rise in WBC 24 h after the infusion of CsA, with no adverse outcomes associated with these findings. There was no significant difference in levels of creatinine, AST and ALT, alkaline phosphatase, hemoglobin or platelets between

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the groups. Additionally, infusion of CsA was associated with increase in mean arterial pressure (MAP) and cerebral perfusion pressure (CPP) for 3 days after the infusion, but none of these increases required therapeutic intervention since because the values of MAP and CPP were still within the range of normal. There were 11 deaths in this trial ranging 8–27 days after the admission with higher rates of mortality within CsA treatment group (n = 9). The causes of deaths were mostly withdrawal of care by the families of the patients. The authors attribute this higher mortality rate to more severe systemic complications suffered by the treatment group patients, but not to the drug effect (Mazzeo et al., 2009). In summary, all studies have similar inclusion criteria and all of them confirmed the safety of CsA for use in TBI patients, which is promising and provides a foothold in the road towards human trials for use of CsA in stroke. However, due to the absence of Phase III trials, it is difficult to predict the future of CsA in TBI and stroke. Although animal trials have shown CsA to be effective in preventing and attenuating brain injury in stroke models, they are in no way yet conclusive and standard treatment regimens are still being tested. For these reasons, it is problematic to predict when it will be possible to commence human studies. 3.5. Adverse effects of CsA Like all immunosuppressants, CsA produces a number of toxic side effects, some of which are quite serious and affect a large number of systems. Nephrotoxicity is seen in 25–75% of patients undergoing treatment with CsA, hypertension has been reported in most cardiac transplantation patients and about 50% of Renal transplant patients, elevated transaminases and bilirubin have also been seen. Other reported reactions included hirsuitism, gingival hypoplasia and seizures (Khan, 2008). A serious adverse reaction seen in patients receiving CsA is posterior reversible encephalopathy syndrome (PRES) (Saeed et al., 2008); this condition is characterized by altered mental status, visual disturbances, and seizures (Akl and Samara, 2010). Neurotoxicity is another adverse side effect observed in patients receiving CsA treatment after organ transplantation. A study conducted by Truwit and colleagues reported three cases of CsAinduced neurotoxicity following organ transplantation. The first patient exhibited CsA neurotoxicity one week after a liver transplant. Severe systemic hypertension accompanied CsA neurotoxicity four weeks after a cardiac transplant in the second patient. Seizures were observed in the third patient one-month after a heart/lung transplant (Truwit et al., 1991). The route of administration of CsA must also be considered. Actis and co-workers demonstrated that 17% of patients receiving IV CsA for the treatment of ulcerative colitis exhibited major toxicity. Conversely, the microemulsion CsA taken orally was as effective as the IV CsA, but these patients did not display serious toxic side effects (Actis et al., 1998). Accordingly, caution must be exercised in designing clinical trials with special emphasis given on the route of administration in view of parenteral and oral CsA differing widely in safety outcomes despite both inducing similar levels of efficacy. Due to its insolubility in water, CsA is prepared for IV injection using a polyoxyethylated castor oil, or cremaphor, and ethyl alcohol. A study conducted by Windebank and colleagues tested rat dorsal root ganglion neurons exposed to the IV preparation of CsA. The dorsal root ganglion neurons that were exposed to the IV preparation exhibited axonal swelling and degeneration. When the neurons were exposed to CsA dissolved in serum there were no adverse effects. However, when the neurons were exposed to 0.1% cremophor, degeneration and axonal swelling was observed. Exposure of 0.001% cremaphor caused demyelination of the axons in vitro (Windebank et al., 1994). It is well known the cremophor produces a myriad of adverse side effects such as hepatotoxicity

(Roman et al., 1989) and nephrotoxicity (Sokol et al., 1990). The literature also suggests that cremaphor contributes to the neurotoxicity exhibited in CsA-treated patients. The optimal pH for CsA efficacy is between 5.5 (Margaritis and Chahal, 1989) and 6.0 (Minones et al., 1994). The altered pH of CsA may also contribute to decreased efficacy. The use of CsA for neuroprotection, as discussed previously, is based on the blockage of the MPTP in the mitochondria of the insulted neural issue. This effect is particularly due to the binding of CsA to the CypD. The drug also exhibits its immunosuppressant properties through calcineurin, which binds to a different site on the surface of the CsA molecule (Borel et al., 1996). These immunosuppressive effects are not favorable when using CsA as a neuroprotective drug, especially with the increased prevalence of chronic illness in patients likely to be receiving such treatment. Experiments aimed at reducing the adverse effects seen with CsA use portrayed promising results. Altering the chemical structure of CsA by replacing amino acid sequences has not produced any calcineurin inhibiting CsA analogs, NIM811 (Melle4-cyclosporin) and UNIL025 (MeAla3EtVal4-cyclosporin) (Waldmeier et al., 2003). Subtle changes at the third and fourth amino acids are the critical binding site for calcineurin; these changes largely abolish the immunosuppressive activity without affecting cyclophilin affinity (Waldmeier et al., 2003). These compounds proved to be effective in blocking the MPT Pand had the same effect as the original drug (Hansson et al., 2004). However, these compounds have only been tested in vitro settings and further in vivo analysis is needed.

4. Conclusion CsA is a well-established immunosuppressive drug that revolutionized post transplantation therapies. Recent research has demonstrated the neuroprotective properties of the drug. CsA blockade of cyclophilin D inhibition of the MPTP has been portrayed to effectively reduce cell death and preserve neuron integrity. These effects have been presented in animal models of stroke and TBI. Human trials are already underway for testing CsA safety and efficacy in TBI. In parallel, promising results seen in animal models demonstrate that CsA does reduce both the histopathological and functional sequelae associated with stroke. Testing for the drug clinically has been only done for use in TBI, and is still limited to Phase II trials. Unfortunately, CsA has shown very high toxicity with a large number of side effects. The parameters for the use of the drug on human subjects are still poorly established. These suggestions are of course preliminary and further testing is needed to establish the efficacy. Safety concerns appear as a major obstacle for CsA clinical application in view of toxicity occurring at a considerably high rate. The idea of developing less toxic CsA analogs that preserve the neuroprotective properties and abolish it toxic and immunosuppressant effects open a new venue for a safer and effective alternative to classic CsA preparations. In summary, the transition of CsA from the laboratory to the clinic will need to overcome serious safety and toxicity concerns. Limited clinical trials in stroke should proceed in tandem with preclinical studies addressing the documented adverse effects of CsA.

Acknowledgements and disclosure This study was supported in part by the Department of Neurosurgery and Brain Repair, the James and Esther King Foundation (1KG01-33966), and the Draper Laboratory, Inc. (09KC-05). C.V.B. is a stockholder and consultant to Maas BiolAB.

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