glycyl-prolyl-glutamic-acid and Hypoxia-Ischemia--Brain

Glycine 2-methyl proline glutamate (G-2mPE) is a proline-modified analogue to the naturally existing N-terminal tripeptide glycine-proline-glutamate that is a cleaved product from insulin-like growth factor-1. G-2mPE is designed to be more enzymatically resistant than glycine-proline-glutamate and to increase its bioavailability. The current study has investigated the protective effects of G-2mPE following hypoxic-ischemic brain injury in the neonatal brain. On postnatal day 7, Wistar rats were exposed to hypoxia-ischemia (HI). HI was induced by unilateral ligation of the left carotid artery followed by hypoxia (7.7% O2, 36 degrees C) for 60 min. The drug treatment started 2 h after the insult, and the pups were given either 1.2 mg/kg (bolus), 1.2 mg/ml once a day for 7 days, or vehicle. The degree of brain damage was determined histochemically by thionin/acid fuchsin staining. G-2mPE's anti-inflammatory properties were investigated by IL-1beta, IL-6, and IL-18 ELISA, and effects on apoptosis by caspase 3 activity. Vascularization was determined immunohistochemically by the total length of isolectin-positive blood vessels. Effect on astrocytosis was also determined in the hippocampus. Animals treated with multiple doses of G-2mPE demonstrated reduced overall brain injury 7 days after HI, particularly in the hippocampus and thalamus compared to vehicle-treated rats. The expression of IL-6 was decreased in G-2mPE-treated animals compared to vehicle-treated pups, and both the capillary length and astrogliosis were increased in the drug-treated animals. There was no effect on caspase 3 activity. This study indicates that peripheral administration of G-2mPE, starting 2 h after a hypoxic-ischemic insult, reduces the degree of brain injury in the immature rat brain. The normalization of IL-6 levels and the promotion of both neovascularization and reactive astrocytosis may be potential mechanisms that underlie its protective effects.

Topics: Animals; Animals, Newborn; Apoptosis; Astrocytes; Birth Injuries; Brain; Caspase 3; Cerebral Arteries; Disease Models, Animal; Drug Administration Schedule; Encephalitis; Gliosis; Hypoxia-Ischemia, Brain; Interleukins; Neovascularization, Physiologic; Nerve Degeneration; Neuroprotective Agents; Oligopeptides; Rats; Rats, Wistar; Time Factors; Treatment Outcome

Insulin-like growth factor-I is a neurotrophic factor and can prevent neurons from ischemic brain injury. However, the large molecular weight and metabolic effects can be problematic in its central delivery. Glycine-proline-glutamate (GPE) is the N-terminal tripeptide of insulin-like growth factor-I, which is naturally cleaved in the plasma and brain tissues. GPE reduces neuronal loss from hypoxic-ischemic brain injury following central administration. Central penetration and the stability of GPE in the plasma and central nervous system were examined in rats using radioimmunoassay and HPLC. GPE was rapidly metabolised in the plasma (8 min) after intraperitoneal administration. Despite having a short half-life in plasma, GPE was detected in the cerebrospinal fluid up to 40 min after intraperitoneal administration. With present of peptidase inhibitors, GPE existed in the brain tissue up to 3 h after intracerebroventricular administration, suggesting a role for peptolysis in its stability. The endopeptidase inhibitors 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) reduced GPE metabolism in the brain tissue while acid peptidase inhibitor pepstatin-A decreased GPE metabolism in the plasma. GPE reduced neuronal loss in the CA1-2 sub-region of the hippocampus given (intraperitoneally) after 30 min of hypoxic-ischemic injury in adult rats, further suggested the effectiveness of GPE central uptake. These results indicated that GPE crosses the blood-CSF and the functional CSF-brain barriers. The longer half-life of GPE in the CNS may be due to its unique enzymatic stability.

Topics: Animals; Brain; Cerebrospinal Fluid; Drug Stability; Hippocampus; Hypoxia-Ischemia, Brain; Insulin-Like Growth Factor I; Kinetics; Leucine; Male; Oligopeptides; Pepstatins; Peritoneum; Protease Inhibitors; Rats; Rats, Wistar

The N-terminal tripeptide of insulin-like growth factor-1, GPE is neuroprotective when given intracerebroventricularly 2 h after hypoxic-ischemic (HI) brain injury in rats. We have now examined whether GPE can cross the blood-brain barrier and exert neuroprotective actions following intravenous administration. Following a single bolus intravenous injection, GPE was rapidly metabolized and cleared from the circulation. The short half-life (<2 min) in blood was subsequently associated with modest and inconsistent neuroprotection. In contrast, potent neuroprotection of GPE was consistently observed in all brain regions examined following 4 h intravenous infusion (12 mg/kg). The neuroprotective effects of GPE after infusion showed a broad effective dose range (1.2-120 mg/kg) and an extended window of treatment to 7-11 h after injury. The central penetration of GPE after intravenous infusion was injury-dependent. GPE also improved long-term somatofunction with a comparable neuronal outcome. GPE reduced both caspase-3-dependent and -independent apoptosis in the hippocampus. Treatment with GPE also inhibited microglial proliferation and prevented the injury-induced loss of astrocytes. In conclusion, the neuroprotective actions of GPE infusion were global, robust and displayed a broad effective dose range and treatment window. GPE's activity included the prevention of neuronal apoptosis, promotion of astrocyte survival and inhibition of microglial proliferation. With injury specific central penetration, GPE has considerable promise as a systemic neuroprotective treatment after acute encephalopathies.

Topics: Animals; Brain Chemistry; Dose-Response Relationship, Drug; Functional Laterality; Half-Life; Hippocampus; Hypoxia-Ischemia, Brain; Immunohistochemistry; In Situ Nick-End Labeling; Infusions, Intravenous; Insulin-Like Growth Factor I; Male; Molecular Weight; Neurons; Neuroprotective Agents; Oligopeptides; Radioimmunoassay; Rats; Rats, Wistar

Insulin growth factor 1 (IGF-1) has an important role in brain development and is strongly expressed during recovery after a hypoxic-ischemic injury. Some of its central actions could be mediated through the N-terminal tripeptide fragment of IGF-1: Gly-Pro-Glu (GPE). The neuroprotective properties of GPE given after a moderate injury in the developing rat brain were evaluated and the binding sites of [(3)H]GPE characterised by autoradiography. After right unilateral injury, GPE or vehicle (V) was injected in the right lateral ventricle (i.c.v.) or in the peritoneal cavity (i.p.) of 21-day-old rats. The percentage of surviving neurons in CA1-2 of the hippocampus was higher in the animals treated with 30 microg of GPE i.c.v. (V: 7.7+/-4.9%, GPE: 26.4+/-7.5%, P=0.02) and 300 microg i.p. (V: 30.2+/-9.1%, GPE: 68.8+/-10.6%, P=0.02) than in animals receiving vehicle. I.p. injection of 300 microg of GPE (V: 78.4+/-7.5%, GPE: 88.4+/-3.2%, P=0.04) was also neuroprotective in the lateral cortex. I.c.v. injection of [(3)H]GPE suggested binding to glial cells in the white matter tracts, the cortex and striatum as opposed to neurons. Although the precise mode of action of GPE is unknown, this study suggests that local administration of GPE is neuroprotective after brain HI injury via glial cells. In addition, systemic administration of GPE showed a more widespread neuroprotective effect. GPE may represent a complementary pathway for central and systemic IGF-1's antiapoptotic effects.

Topics: Animals; Brain; Cell Survival; Cerebrovascular Circulation; Female; Hypoxia-Ischemia, Brain; Hypoxia, Brain; Injections, Intraperitoneal; Injections, Intraventricular; Insulin-Like Growth Factor I; Male; Neurons; Neuroprotective Agents; Oligopeptides; Rats; Rats, Wistar