The role for IGF-1-derived small neuropeptides as a therapeutic target for neurological disorders.

Review

The role for IGF-1-derived small neuropeptides as a therapeutic target for neurological disorders 1.

Introduction

2.

Glycine-proline-glutamate

3.

Cyclic Gly-Pro and its analogue,

Jian Guan†, Paul Harris, Margaret Brimble, Yang Lei, Jun Lu, Yang Yang & Alistair J Gunn †

The University of Auckland, Liggins Institute, Auckland, New Zealand

Expert Opin. Ther. Targets Downloaded from informahealthcare.com by Kainan University on 04/10/15 For personal use only.

cyclic Gly-aallyl-Pro (NNZ2591) 4.

Mechanisms of neuroprotection

5.

Expert opinion

Introduction: Exogenous IGF-1 protects the brain from ischemic injury and improves function. However, its clinical application to neurological disorders is limited by its large molecular size, poor central uptake and mitogenic potential. Areas covered: In this review, the authors have discussed the efficacy, pharmacokinetics and mechanisms of IGF-1 derivatives on protecting acute brain injury, preventing memory impairment and improving recovery from neurological degenerative conditions evaluated in various animal models. We have included natural metabolites of IGF-1, glycine-proline-glutamate (GPE), cleaved from N-terminal IGF-1 and cyclic glycine-proline (cGP) as well as the structural analogues of GPE and cGP, glycine-2-methyl-proline-glutamate and cyclo-L-glycyl-L-2-allylproline, respectively. In addition, the regulatory role for cGP in bioavailability of IGF-1 has also been discussed. Expert opinion: These small neuropeptides provide effective neuroprotection by offering an improved pharmacokinetic profile and more practical route of administration compared with IGF-1 administration. Developing modified neuropeptides to overcome the limitations of their endogenous counterparts represents a novel strategy of pharmaceutical discovery for neurological disorders. The mechanism of action may involve a regulation of IGF-1 bioavailability. Keywords: cyclic glycine-proline, glycine-proline-glutamate, IGF-1, ischemic brain injury, Parkinson’s disease, rat Expert Opin. Ther. Targets [Early Online]

1.

Introduction

IGF-1 and its family IGF-1 plays an essential role in normal brain development and metabolism [1], and recovery from injury [2]. IGF-1 function is mediated by binding to specific IGF-1 receptors that initiate downstream signaling pathways [3,4]. IGF-1 homeostasis is dynamically regulated by reversible binding to circulating and tissue-associated IGF-1 binding proteins (IGFBPs) [5]. Poor recovery from ischemic brain injuries and neurodegenerative conditions have been associated with reduced IGF-1 bioactivity [2]. 1.1

Endogenous response of IGF-1 to brain injury Circulating IGF-1 accumulates in the blood vessels of damaged brain regions within a few hours after hypoxic-ischemic (HI) injury [6]. Glial cells show increased production of IGF-1 for days after injury [6]. However, immediately after HI IGF-1 gene expression is reduced [7], there is reduced expression of intracellular pathways activated by IGF-1 such as phosphorylated Akt [8]. Furthermore, the mRNA transcripts encoding IGF-1 receptors either remain the same or decrease after brain injury [6,9], 1.2

10.1517/14728222.2015.1010514 © 2015 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

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Article highlights. . . .

IGF-1 and its metabolites are capable of preventing brain injury and promoting brain recovery. Compared with IGF-1 its metabolites have advanced pharmacokinetics and dynamics. The observation of cGP on regulating bioavailability of IGF-1 suggest a deficit specific function of IGF-1 metabolites.


This box summarizes key points contained in the article.

and the IGFBPs show different temporal and spatial expression depending on the degree of injury. These data suggest that in some types of injury there may be insufficient endogenous IGF-1 to facilitate effective brain recovery [10]. IGF-1 protein has been immunohistochemically shown to be associated with reactive glia rather than neurons after HI in the developing brain [11,12]. Similar findings have been reported by several laboratories in immature [7,13] and adult [14] rats. In addition to changes after acute brain injury, plasma concentrations of IGF-1 decline with aging and IGF-1 deficiency is closely related to the onset and progress of agerelated neurodegenerative conditions [15]. Taken together, these findings raise the possibility that treatment with IGF-1 might help improve neurological recovery and function. IGF-1 protects brain from ischemic injuries Central administration of IGF-1 prevents both necrotic and apoptotic neuron death after HI brain injury in the adult rat [16] and fetal sheep [17,18]. The treatment effects are dose dependent, for example, there is a U-shaped dose response for neuroprotection after 30 min of cerebral ischemia in fetal sheep [16,17]. Similarly, intracerebroventricular (ICV) infusion of 3 µg of IGF-1 started 90 min after severe cerebral ischemia in near-term fetal sheep prevented loss of oligodendrocytes and subsequent demyelination in the parasagittal cerebral cortex [18]. However, adding a continuous infusion of IGF-1 (1 or 3 µg) for 24 h after the initial short (1 h) infusion of IGF-1 (3 µg) did not further improve neuroprotection compared with a short infusion alone, showing that exogenous IGF-1 is acting on relatively early events after HI [18]. Neuronal loss after brain injury can continue in a progressive, ‘tertiary’ phase over many months after HI [19]. Thus, early protection by some compounds may not be maintained over time [20], and long-term assessment of functional recovery is essential to confirm treatment efficacy. Reassuringly, in rats, treatment with IGF-1 improved long-term somatomotorsensory function, as measured by the bilateral tactile test, and quantitative histological changes 3 weeks after HI [10]. A key practical issue in drug discovery for acute brain injury is that treatment of patients in a timely manner is a formidable problem [20]. For example, despite the proven efficacy of anticoagulants after acute ischemic stroke, the great majority of patients are not able to be enrolled within the 3 h treatment 1.3

2

window [21]. The effective window of opportunity for IGF-1 is about 2 h after HI injury, and it was not neuroprotective when infused 6 h after HI [22]. This relatively short window of opportunity is related to the timing of evolution of post-ischemic neuronal injury. Interestingly, the window of opportunity for treatment with IGF-1 can be significantly prolonged to 6 h if rats are recovered in a cooled environment for just the first 2 h of recovery. Thus, injury-induced brief cerebral cooling may prolong the window of opportunity for other therapies [22]. IGF-1 is reported to have poor uptake into the brain after peripheral administration [23]. However, neuroprotection by IGF-1 after peripheral administration has been reported in several studies [24,25], and further in rats it is taken up effectively after intranasal treatment [26]. Rather the major concerns for use as a treatment are the significant metabolic effects of IGF-1, such as hypoglycemia, and its potential mitogenic effects [27]. 2.

Glycine-proline-glutamate

Discovery of glycine-proline-glutamate Unbound IGF-1 enzymatically metabolizes to des-N-(1-3)IGF-1 (des-IGF-1) and the N-terminal tripeptide of IGF-1, glycine-proline-glutamate (GPE) [28,29]. GPE is a key binding site for IGF-1 to interact with its IGFBPs; thus after cleavage the binding of des-IGF-1 is largely reduced, GPE was generally believed to be a non-bioactive by product. IGF-1 is metabolized by an acid protease in plasma and brain tissues, and endogenous cleavage is enhanced in an acidic environment [29-31]. Bioactivity of GPE was first reported in 1989 [32]. GPE was shown to stimulate both dopamine and acetylcholine release in brain slice culture without interacting with the IGF-1 receptor [33]. In this review, we discuss the discovery, pharmacokinetics and dynamics and mechanism of IGF-1 and its derivatives, as well as the potential for future research to establish IGF-1 as a therapeutic target for neurological conditions. 2.1

Neuroprotection with GPE Saura et al. showed in an organotypic culture that GPE dosedependently protected hippocampal neurons from NMDAinduced neuronal toxicity [34]. The pattern of neuroprotection with GPE appeared to be similar to that by MK801, a potent and non-competitive NMDA receptor antagonist. However, despite the previously reported NMDA-mediated effects of GPE [35,36], GPE showed only weak binding in the dentate gyrus, where NMDA receptors are strongly expressed [34]. Consistent with this, Alonso De Diego et al. demonstrated that neuroprotection with GPE analogues was not correlated with glutamate receptor binding [37], suggesting that protection was mediated through other mechanisms. The neuroprotective effects of GPE in vivo were first evaluated using doses equimolar to neuroprotective doses of IGF-1. A single dose of GPE, given centrally 2 h after HI 2.2

Expert Opin. Ther. Targets (2015) 19(6)


IGF-1 as a therapeutic target for neurological disorders

injury reduced brain injury in adult rats [38]. Similarly, GPE also reduced HI brain injury in infant rats, although interestingly a much higher dose was required for protection (30 µg) than adult rats (3 µg) after central administration [39]. In view of the ability of GPE to promote acetylcholine release, the authors also examined the possibility of selective protection by GPE in different neuronal phenotypes, as well as its potential effects in promoting neuroplasticity [38]. ICV infusion of 3 µg GPE after HI in adult rats selectively attenuated loss of choline acetyltransferase-positive+ cholinergic neurons and glutamate acid decarboxylase (GAD)- and somatostatinimmunopositive GABA interneurons, but not parvalbumin expressing neurons, even though GAD and parvalbumin are typically co-expressed in the same population of GABAergic neurons. This finding suggests that GPE modulated cell function as well as survival. It is also notable that GPE increased the expression of neuronal nitric oxide synthase in the non-injured hemispheres compared with the vehicle-treated control. These selective effects on neuronal phenotypes suggested the possibility that GPE may have promoted neuronal plasticity. Like most small, endogenous peptides, GPE is not enzymatically stable. The half-life of GPE in plasma is < 2 min after single-bolus intravenous injection and < 4 min after intraperitoneal administration to normal adult Wistar rats [40,41]. Endogenous proteases appear to have a role in GPE metabolism, as Bestatin, a protease inhibitor, reduced GPE metabolism in plasma and brain tissues [41]. Protease activity in the brain ex vivo appears to be lower than in plasma, which may explain the longer half-life of GPE (> 30 min) in cerebrospinal fluid (CSF) and brain tissues than after peripheral injection [40,41]. Thus, continuous intravenous administration of native GPE is essential to maintain efficacious plasma levels and achieve reliable treatment effects. GPE shows an injury-dependent ability to cross the blood--brain barrier (BBB), with much greater uptake after intravenous administration into the brain after HI than in healthy rats [19]. The hydrophilic nature of GPE likely limits its central penetration in the normal brain, whereas injuryactivated MMP-2 and -9 can disrupt the basement membranes, thus increasing the permeability of the BBB [42,43]. Neuroprotection with GPE after continuous intravenous infusion is robust with a broad effective dose range and a window of opportunity of up to 7 h after HI injury, and offers long-term protection and recovery in function [19]. Although the rapid plasma clearance of GPE noted above would limit its suitability for treating chronic neurological conditions, this could be favorable for treating acute neurological conditions, since adverse effects associated with drug accumulation can be easily minimized [19]. Continuous delivery of GPE for 7 days also reduced neuronal death in the hippocampus after cardiac arrest in adult rats [44]. The majority of stroke patients are over 65 years old. Aging affects systemic physiology, brain morphology and biology [45,46] and may alter how the brain responds both to ischemic brain injury and to potential treatments [47]. Therefore,

we examined whether there were age-related effects of GPE treatment after microsphere-induced embolic brain injury in comparison with young adult rats. A unilateral nonreperfusion stroke was induced by infusion non-radioactive microspheres into the right internal carotid artery in young adult (3 -- 4 months) and aged (16 -- 17 months) male rats. GPE (12 mg/kg) or the vehicle was then infused intravenously over 1 h, starting 3 h after embolic injury. Encouragingly, GPE treatments significantly and similarly reduced the degree of brain injury in both young adult and aged rats, showing that the effect of GPE is not modified by age. This treatment effect after embolic injury was independent of blood glucose, core temperature, oxygen saturation and heart rate [48]. Effect of GPE on neurodegeneration We used an animal model of 6-hydroxydopamine (6-OHDA)induced nigrostriatal depletion to examine whether GPE had specific treatment effects on dopamine neurons. A single dose of GPE (3 µg) given centrally 2 h after the lesion prevented loss of tyrosine hydroxylase (TH) immunopositive neurons in the substantia nigra compared with the vehicle-treated group. GPE treatment may also promote distal neurotransmission, as TH immunoreactivity in the striatum was also strongly increased compared with the vehicle-treated group [49]. Furthermore, administration of GPE reduced apomorphineinduced rotations and attenuated long-term forelimb akinesia after 6-OHDA injection [50,51]. The mechanism underlying the neuroprotection of GPE may be related to covalent binding of GPE to L-DOPA [52]. In addition to these effects in animal models of Parkinson’s disease, beneficial effects of GPE have also been reported in animal models of Huntington’s disease [53], Alzheimer’s disease [54] and depression [55]. 2.3

Neuroprotective effects of glycine 2-methyl proline glutamate (NNZ2566)

2.4

To overcome the instability of GPE in plasma, a GPE analogue was generated by adding a methyl group to the proline ring of GPE, to increase resistance to enzymatic degradation [56]. The half-life of this compound, glycine 2-methyl proline glutamate (G-2mPE), in the plasma was indeed significantly prolonged compared with GPE [57]. The neuroprotective effects of this analogue have been reported subsequently in both adult and neonatal rat models of ischemic and HI brain injury [58,59]. Zhao et al. examined the treatment effect of GPE, G-2mPE and their combination with caffeinol, a mixture of caffeine and ethanol, on neurological deficit after middle cerebral artery occlusion. Continuous intravenous infusion of either GPE (0.3 mg/kg/h 4 h) or G-2mPE at a 10-fold lower dose significantly reduced the neurological deficit compared with the vehicle-treated rats [58]. The authors also found an additive treatment effect between G-2mPE and caffeinol, but not between GPE and caffeinol [58]. More recently, Svedin et al. reported that in post-natal day 7 rats daily treatment with G-2mPE given intraperitoneally for


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γ H NOE H

β

H N

δ

H N

CO2H N

N

O

O α NH2

CO2H

O

O

CO2H

CO2H NH2

GPE (trans) 80%

GPE (cis) 20%


Figure 1. The trans and cis isoform of glycine-proline-glutamate (GPE).

7 days after HI improved neuronal outcome 7 days after HI injury [59]. The treatment effect was significantly less when a single dose was given 2 h after HI, consistent with ongoing evolution of cell death well after the original insult. As part of preclinical development of the analogue, its protective effects were also evaluated in animal models of traumatic brain injury and ischemia-induced seizures [60-62]. These studies suggested that protection was associated with antiinflammatory and anti-apoptotic effects [63].

Cyclic Gly-Pro and its analogue, cyclic Gly-aallyl-Pro (NNZ2591) 3.

Cyclic Gly-Pro Bioactivity of cyclic-Gly-Pro (cGP) has been demonstrated as an endogenous nootropic factor [64], which can be formed through GPE metabolism [65]. Our unpublished data from NMR spectroscopy demonstrated that GPE exists as a 80:20 trans:cis isomeric mixture around the prolyl-glycine bond (Figure 1). In small proline-containing peptides, the chemical shift difference between the g and b carbons of the proline residue is generally larger in the case of the cis isomer (ca. 10 ppm) when compared with the trans form (ca. 5 ppm) [66]. In the case of GPE, 13C NMR spectroscopy recorded in heavy water revealed this difference to be 5.1 ppm, thus determining the major form to be in the trans configuration [67]. In addition, there was a strong Nuclear Overhauser Effect observed in the 1H spectrum of GPE between the glycine a-protons and the d-protons that can only occur for the trans isomer [67]. The differences in prolyl-glycine binding between the trans and cis isoforms may determine the formation of cGP after the enzymatic cleavage of the glutamate. Such cyclic structures may render a molecule resistant to enzymatic breakdown and also be more lipophilic for better central uptake. 3.1

Neuroprotection of cGP and its analogue after ischemic brain injury

3.2

Both cGP and its structure analogue cGly-2allyl-Pro (cG-2allylP, NNZ2591), have been shown to protect brain from ischemic injury in a dose-dependent manner [68]. It is different from the tripeptide in that the central uptake of cG-2allylP is not injury dependent, so that in normal control rats the 4

amounts of free cG-2allylP detected in CSF were similar to those found in plasma [68]. Although the level of cG-2allylP in blood fell to half of the initial value 2 h after a single intravenous dose, the CSF level was stable from 0.5 to 2 h after a bolus injection [68]. CSF is produced continuously, and turns over approximately 1 h in rats. It is the constant reabsorption of CSF back into plasma that is largely responsible for drug elimination from the CNS [19,69]. Thus, the maintained CSF level of cG-2allylP for 2 h suggests a sustained central transfer of cG-2allylP from plasma and thus that central uptake of cG-2allylP compensates for the drug’s removal through CSF absorption. cG-2allylP is still detectable in CSF 6 h after administration. An in vitro albumin-binding assay suggests that there is likely a process of protein binding and releasing of cG-2allylP in the plasma, which may alter the pharmacokinetics of cG-2allylP by influencing the level of free compound in the plasma [68]. Intriguingly, neuroprotection with cG-2allylP shows complex dose-dependency after central administration, with higher doses being ineffective [68]. In contrast, after peripheral administration the three different doses of cG-2allylP tested were all effective. It is possible that the possible protein binding-releasing process noted above may provide a sustained release depot to stabilize levels of the free compound in plasma [70]. Furthermore, whereas a single treatment resulted in modest histological improvement, repeated treatment with cG-2allylP was associated with almost complete neuroprotection after 9 weeks recovery [68]. Supporting this, repeated treatment with cG-2allylP also prevents HI injury-induced somatosensory-motor deficit [68].

Other neurological conditions Two weeks of administration of cG-2allylP after the onset of 6-OHDA-induced motor deficits improved functional recovery for up to 12 weeks. The extent of recovery was independent of the dopamine depletion in rats, possibly due to enhanced neurogenesis [71]. A role for cG-2allylP in neuroplasticity was also suggested by improved memory in a rat model with scopolamine-induced acute memory impairment. This improvement was associated with evidence of improved cholinergic neurotransmission [72]. 3.3



4.

Mechanisms of neuroprotection

Anti-apoptosis and anti-inflammation Neuroprotection with IGF-1 derivatives are clearly involved in preventing necrotic and apoptotic neuronal death as shown by reduced area of tissue infarction and numbers of cleaved caspase-3-positive neurons [60-63,73]. Inhibition of injuryinduced inflammation responses has also been suggested by the association of neuroprotection with these derivatives, and subsequently reduced levels of pro-inflammatory cytokines (IL-6 and TNF-a) and numbers of reactive microglia [60-63]. Furthermore, there was evidence of improved vascular remodeling, probably through either elevation in and/or protection of activation of astrocytes [73].


4.1

Vascular protection of cGP was mediated via activation of IGF-1 receptors

4.2

While the neuroprotective effect of IGF-1 is clearly mediated by IGF-1 receptors [16], the mode of action of GPE and cGP remains unclear as GPE does not bind to the IGF-1 receptor [32,34,68,73]. Despite the failure to identify a specific receptor, the neuroprotective effects of GPE and cGP after central (ICV) administration are clearly dose-dependent, consistent with receptor-mediated pharmacodynamics [73]. It is also known that the neuroprotective effects of IGF-1 [74] and GPE [48,59] involve reduced vascular damage and improved vascular remodeling. Thus, the potential role of changes in the IGF-1 receptor in vascular protection with cGP was investigated. HI brain injury is associated with unilateral brain damage in the hemisphere ipsilateral to carotid artery ligation [59], with more subtle contralateral histological changes mediated by brief hypoxia alone [38]. HI injury was associated with the loss of capillaries on the ipsilateral (ligated) hemisphere. Treatment with cGP (0.2 µg ICV) that partially reduced neuronal damage was associated with complete preservation of capillary density [68]. These vascular effects of cGP may contribute to its neuroprotective properties [68] as a preserved vascular network is important for long-term function and survival of brain tissues [59,75]. The potential role for IGF-1 in this vascular protection was then evaluated by assessing the expression of both inactivated and phosphorylated IGF-1 receptors in the capillaries after cGP treatment [76]. The IGF-1 receptors appeared to be localized on the pericytes, the capillary cells involved in angiogenesis. There was loss of IGF-1 receptor-positive capillaries, suggesting that HI injury compromised IGF-1-mediated vascular remodeling [76]. Treatment with cGP restored IGF-1 receptor-associated angiogenic capillaries. These data suggest that cGP promotes endogenous IGF-1-induced vascular remodeling, when levels of endogenous IGF-1 are insufficient to prevent neuronal and vascular damage. Furthermore, this suggested the hypothesis that the protective effects of cGP could be mediated indirectly through IGF-1. Supporting

this hypothesis, in vitro treating human endothelial cells with cGP stimulated IGF-1-induced cell growth after either serum withdrawal or IGF-1 receptor knockdown [76]. Thus, these cGP-associated effects on cell growth were associated with expression of IGF-1 receptors. Unexpectedly, we also found an inhibitory effect of cGP when IGF-1-induced cell growth was increased by either overexpression of IGF-1 receptors or blockade of IGFBP-3. Similarly, inhibitory effects of cGP were also demonstrated in a mice model of lymphoma where cGP prevented IGF-1-mediated tumor growth [76]. Using an in vitro peptide--peptide interaction assay and HPLC analysis [76], cGP treatment was found to release IGF-1 from the reversible IGF-1/IGFBP-3 binding, but only when the amount of free IGF-1 was relatively lower than cGP, whereas cGP treatment reduced free IGF-1 when IGF-1/cGP ratio was higher. In contrast, cGP did not alter IGF-1 binding to IGFBP-3 when the concentrations of cGP and IGF-1 were similar. These data are unlikely to completely explain the complex biological interaction of IGF-1 and cGP to IGFBPs, but suggest that the relative concentrations of cGP and IGF-1 do influence IGF-1 function by altering binding of IGF-1 to IGFBP-3. As the N-terminal tripeptide of IGF-1, GPE makes a major contribution to the binding of IGF-1 to its IGFBPs [32], since des-IGF-1 has low affinity for IGFBPs [5]. IGFBPs can inhibit the formation of GPE by preventing free IGF-1 from being metabolized [35]. Given the fact that GPE is enzymatically unstable [41], does not exist in material amounts in tissues [32] and shares many biological characteristics with cGP [68], as well as being transformed to cGP, which is much more stable, it is possible that in normal tissues cGP may interact with IGFBPs and, thereby contribute to regulation of IGF-1 activity. We propose that reversible binding of IGF-1 to IGFBPs maintains physiological IGF-1 metabolism and that the release of the enzymatic product of free IGF-1, cGP, helps to maintain and/or restore the equilibrium between free and bound IGF-1 by altering binding of IGF-1 to IGFBPs. If this is correct, then cGP would be a significant local factor in autoregulation of IGF-1 action, and may have pharmacological significance. This hypothesis suggests that when IGF-1 activity is relatively insufficient, additional cGP may competitively displace IGF-1 from IGFBPs to increase the availability of free IGF-1 (Figure 2). Even though the mechanisms underlying any interactions between cGP-IGFBP-3 have not been elucidated, these findings suggest that exogenous administration of cGP is able to modulate IGF-1/IGFBP-3 binding and so restore IGF-1 bioactivity and may underlie the neuroprotective effects of cGP and other IGF-1 metabolites, including GPE and their analogues. Supporting inhibitory effects of cGP in vivo, we found that the administration of cGP prevented IGF-1-dependent tumor growth in two different models of cancer in mice [76] (unpublished data).


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A.

B. Increase Free IGF-1 Reduce IGF-1 binding to IGFBP-3

Enzymatic break done

Enzymatic break done

cGP

Insufficient Free IGF-1

IGF-1 binding to IGFBP-3

Free IGF-1

Normal cGP/IGF-1 ratio Add cGP

Ratio of cGP IGF-1


Increase cGP/IGF-1 ratio C. Normalise Free IGF-1 Elevated Free IGF-1

Increase IGF-1 binding to IGFBP-3

Reduced Enzymatic break done? Reduced cGP/IGF-1 ratio Add cGP Normalise cGP/IGF-1 ratio

Figure 2. Flowcharts showing dyn amic regulatory axis of cGP on IGF-1 function under physiological condition (A). IGF-1 function is insufficient during injury recovery (B). IGF-1 activity elevated due to insufficient enzymatic break down of IGF-1 (C). A. Balanced regulatory axis in which ratio of cGP/IGF-1 controls IGF-1 dynamically bioavailability under physiological conditions. B. Physiological function of IGF-1 may be relatively insufficient due to increased demand from tissue repairing during the recovery from brain injury. Administration of cGP increases cGP/IGF-1 ratio resulting in the reduction of IGF-1/IGFBP-3 bound, thus increasing the bioavailability of the IGF-1 [76]. C. The changes of the axis when IGF-1 function is elevated due to the increase of IGF-1 bioavailability. The cause of increase of free IGF-1 is unknown. One potential cause may be insufficient enzymatic activity to cleave IGF-1 into cGP, leading to a lower ratio of cGP/IGF-1. Administration of cGP can restore the physiological ratio of cGP/IGF-1 leading to an increase in IGF-1/IGFBP-3 bound, thus normalizing the bioavailability of IGF-1 [76].

5.

Expert opinion

The induction of endogenous IGF-1 after brain injury suggested that it plays a role in preventing brain injury. Indeed, treatment with exogenous IGF-1 after brain injury is protective and leads to improved long-term neurological function. However, there is limited potential for IGF-1 to be used for treating neurological conditions in view of its poor central uptake. Its mitogenic effects are also a major roadblock for clinical application. Compared with IGF-1, GPE is a very small molecule, and does not interact directly with IGF-1 receptors, thus it shows effective central penetration without the potential side effect of stimulating cell growth. However, the major disadvantage of GPE for clinical use is that it is enzymatically unstable. The structural analogues of GPE showed greater 6

efficacy in protecting brains from acute injury and improving brain recovery, presumptively due to their improved enzymatic stability and central delivery. Compared with GPE, which is a transient intermediate during IGF-1 metabolism, cGP, the final metabolite, is resistant to enzymatic degradation, and has a favorable pharmacokinetic profile. Structural modification of cGP also moderately improved pharmacodynamics. In addition to the finding that treatment with these compounds can reduce histological brain damage and improve brain function, our knowledge of the specific mechanisms of action is still limited to the observation of secondary effects such as inhibition of inflammation, which in principle may be a consequence of reduced brain damage. Even though the specific mechanism of action of these peptides is still being elucidated, there is increasing evidence that the effects of cGP and GPE, at least in part, reflect regulation




of IGF-1 activity. Potentially, this may be mediated by increasing or reducing the bioavailability of IGF-1, by altering the binding of IGF-1 to its binding proteins. This hypothesis is based on experimental observations that cGP can either promote or inhibit IGF-1-induced bioactivity when it is relatively deficient or elevated, respectively [76]. This recent finding may explain the apparently injury-dependent protective effects of the analogues and conversely, the lack of neurological improvement seen in a clinical trial of GPE in which the subjects had only mild or no neurological deficits. The precise regulatory mechanism is still being investigated, for example, studies are needed to confirm physical binding of cGP and/or IGF-1 to the six different IGF-binding proteins, and to provide clinical evidence for the regulatory effects of cGP on IGF-1 actions. Based on > 20 years of research into IGF-1-derived small peptides, there are currently two FDA-approved clinical trials testing the analogues in the USA. Whether the proposed mechanism of regulation of IGF-1 bioavailability applies to these analogues being developed by Neuren Pharmaceuticals (New York, NY, USA) needs to be evaluated. Further investigation into the regulatory mechanisms may open up new opportunities for their clinical applications to many

conditions associated with impaired homeostasis of IGF-1, for example, the dysfunction of energy metabolism, cancer and the poor growth after birth in infants with growth restriction at birth. Treatments that either block or stimulate IGF-1 function through manipulating the receptor, ligands and downstream signaling pathways have been used to treat medical conditions with dysfunction of IGF-1, for example, cancer. A major issue for the clinical translation of these treatments is the potential for adverse effects from impairment or exaggeration of the normal physiological functions of IGF-1. In contrast, the evidence discussed above suggests that exogenous cGP may only alter IGF-1 function under pathological conditions, without disturbing normal physiology.

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

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Affiliation

Jian Guan†1,2, Paul Harris3, Margaret Brimble2,3, Yang Lei4,5, Jun Lu4, Yang Yang5 & Alistair J Gunn2,7 † Author for correspondence 1 University of Auckland, Liggins Institute, Private Bag 92019, Auckland, New Zealand Tel: +64 93 737 599 ext. 86134; Fax: +64 93 082 385; E-mail: [email protected] 2 University of Auckland, Centre for Brain Research, Faculty of Medicine and Health Sciences, Auckland, New Zealand 3 University of Auckland, School of Chemistry, Department of Medicinal Chemistry, Auckland, New Zealand 4 Northeast Normal University, Institute of Genetics and Cytology, Changchuen, China 5 Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China 6 Wuhan University of Technology, Affiliated Tian-you Hospital, Department of Endocrine and Metabolism, Wuhan, China 7 University of Auckland, School of Medical Sciences, Department of Physiology, Auckland, New Zealand

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