John Randall Fawcett

Intranasal administration of insulin-like growth factor-I bypasses the blood-brain barrier and protects against focal cerebral ischemic damage

BACKGROUND Insulin-like growth factor-I (IGF-I) has been shown to protect against stroke in rats when administered intracerebroventricularly. However, this invasive method of administration is not practical for the large number of individuals who require treatment for stroke. Intranasal (IN) delivery offers a noninvasive method of bypassing the blood-brain barrier (BBB) to deliver IGF-I and other neurotrophic factors to the brain. Here, we demonstrate for the first time the therapeutic benefit of IN IGF-1 in rats following middle cerebral artery occlusion (MCAO). METHODS A blinded, vehicle-controlled study of IN IGF-I was performed using the intraluminal suture occlusion model. Rats were randomly divided into vehicle-control, 37.5 and 150 microg IGF-I-treated groups. Treatments occurred at 10 min after onset of 2 h of MCAO, and then 24 and 48 h later. Four neurologic behavioral tests were performed 4, 24, 48 and 72 h after the onset of MCAO. Corrected infarct volumes were evaluated 72 h after the onset of MCAO. RESULTS Treatment with the 150 microg IGF-I significantly reduced the infarct volume by 63% vs. control (p=0.004), and improved all the neurologic deficit tests of motor, sensory, reflex and vestibulomotor functions (p<0.01). However, the 37.5 microg dose of IGF-I was ineffective. CONCLUSION While IGF-I does not cross the BBB efficiently, it can be delivered to the brain directly from the nasal cavity following IN administration, bypassing the BBB. IN IGF-I markedly reduced infarct volume and improved neurologic function following focal cerebral ischemia. This noninvasive, simple and cost-effective method is a potential treatment for stroke.

Delivery of Nerve Growth Factor to the Brain via the Olfactory Pathway

Purpose: To assess the potential of delivering nerve growth factor (NGF) to the brain along the olfactory neural pathway for the treatment of Alzheimers disease. Methods: Recombinant human NGF (rhNGF) was given as nose drops to anesthetized rats. The rhNGF concentrations in the brain were determined by enzyme-linked immunosorbent assay (ELISA). Results: Following olfactory administration, rhNGF reached the brain within an hour, achieving a concentration of 3400 pM in the olfactory bulb, 660–2200 pM in other brain regions and, 240 pM and 180 pM in the hippocampus and the amygdala, respectively. In contrast, little or no rhNGF was found in the brain following intravenous administration. Conclusions: A significant amount of rhNGF can be delivered to the brain via the olfactory pathway. The detection of rhNGF by ELISA indicates that rhNGF is delivered to the brain relatively intact. The rapid appearance of rhNGF in the brain suggests that it may be transported by an extraneuronal route into the brain via intercellular clefts in the olfactory epithelium. Further work to clarify the transport mechanism is underway. The olfactory pathway is a promising, non-invasive route for drug delivery to the brain, which has potential for the treatment of neurodegenerative diseases including Alzheimers disease.

Delivery of 125I-NGF to the Brain via the Olfactory Route

AbstractThe blood-brain barrier presents a major problem in the administration and testing of neurotropins as it prevents a sufficient concentration of these potential therapeutic agents from reaching the target areas of the human brain. The olfactory neuroepithelium is the only area of the body in which an extension of the central nervous system comes into direct contact with the environment. Following intranasal administration of 125I-labeled nerve growth factor (125I-NGF), radiolabel appeared rapidly in the olfactory bulb and other brain regions. Radiolabel accumulation in the olfactory bulb of the brain was a linear function of the intranasal dose and of the radiolabel concentration in the olfactory epithelium. Concentration of radiolabel in the olfactory bulb and brain with intranasal administration, but not with intravenous administration, suggests direct transport of label into the brain along the olfactory route following intranasal administration. The rapid appearance of label in the olfactory bu...

Non-invasive intranasal insulin-like growth factor-I reduces infarct volume and improves neurologic function in rats following middle cerebral artery occlusion.

Insulin-like growth factor-I (IGF-I) has been proposed as a treatment for stroke. However, it does not efficiently cross the blood-brain barrier (BBB). Intracerebroventricular injection of IGF-I has been shown to offer protection against cerebral ischemic damage in rats although this invasive method of administration may not be practical in humans. Non-invasive intranasal (IN) delivery of IGF-I to the brain is a promising alternative. We have assessed the therapeutic effect of IN IGF-I in rats following middle cerebral artery occlusion (MCAO). Treatment was initiated 10 min after the onset of MCAO and then again 24 and 48 h later. Intranasal dosing of 75 microg IGF-1 (225 microg total IGF-I over 48 h) significantly reduced corrected infarct volumes by 60% vs. control (P<0.01) and hemispheric swelling by 45.6% vs. control (P<0.05). Neurologic function, assessed by the postural reflex, flexor response and adhesive tape tests, was also improved by IN IGF-I as compared to control. Our study indicates IN delivery of IGF-1 holds significant promise as a non-invasive and efficacious method of bypassing the BBB for the treatment of stroke.

Inactivation of the human brain muscarinic acetylcholine receptor by oxidative damage catalyzed by a low molecular weight endogenous inhibitor from Alzheimer's brain is prevented by pyrophosphate analogs, bioflavonoids and other antioxidants.

Oxidative stress has been implicated as a contributing factor to neurodegeneration in Alzheimers disease. An endogenous, low molecular weight (LMW) inhibitor from Alzheimers brain inactivates the human brain muscarinic acetylcholine receptor (mAChR). The inhibitor prevents agonist and antagonist binding to the mAChR as assessed by radioligand binding studies. The LMW endogenous inhibitor, which has components with molecular weights between 100 and 1000 Da, requires dissolved oxygen and glutathione. Prevention of inactivation of the mAChR with peroxidase suggests that the LMW endogenous inhibitor generates peroxide. Heme, previously shown to be present in the LMW endogenous inhibitor, also inactivates the mAChR in the presence of peroxide. Free radical damage to the muscarinic receptor by the endogenous inhibitor can be prevented through the use of naturally occurring antioxidants including bilirubin, biliverdin, carnosol, myricetin and quericetin. In addition, pyrophosphate, imidodiphosphate, bisphosphonates and related compounds also protect the muscarinic receptor from free radical damage. Inactivation of the mAChR by the LMW endogenous inhibitor is likely to be a factor in the continual decline of Alzheimers patients, even those taking acetylcholinesterase inhibitors. Natural antioxidants and pyrophosphate analogs may improve the effectiveness of acetylcholinesterase inhibitors and prove useful in the treatment and prevention of Alzheimers disease since the muscarinic acetylcholine receptor is required for memory, and decreased cholinergic function is a critical deficit in Alzheimers disease.

Anandamides inhibit binding to the muscarinic acetylcholine receptor

Loss of memory and cholinergic transmission are associated with both Alzheimer’s disease (AD) and marijuana use. The human brain muscarinic acetylcholine receptor (mAChR), which is involved in memory function and is inhibited by arachidonic acid, is also inhibited by anandamides. Two agonists of the cannabinoid receptor derived from arachidonic acid, anandamide (AEA) and R-methanandamide, inhibit ligand binding to the mAChR. Binding of the mAChR antagonist [3H]quinuclidinyl benzilate ([3H]QNB) is inhibited up to 89% by AEA (half-maximal inhibition at 50 µM). Binding of the more polar antagonist [N-methyl-3H] scopolamine ([3H]NMS) is inhibited by AEA up to 76% (half-maximal inhibition at 44 µM). R-methanandamide inhibits more than 90% of both [3H]QNB binding (I50=34 µM) and [3H]NMS binding (I50=15 µM) to the mAChR. Both AEA and R-methanandamide stimulate mAChR binding of the agonist [3H]oxotremorine-M at low concentrations (25–75 µM), but significantly inhibit agonist binding at higher concentrations (I50=150 µM). The cannabinoid antagonist SR141716A did not alter AEA or R-methanandamide inhibition of [3H]NMS binding to the mAChR, even at concentrations as high as 1 µM. Further, the cannabinoid agonist WIN 55212-2 does not alter antagonist binding to the mAChR. This demonstrates that mAChR inhibition by the anandamides is not mediated by the cannabinoid receptor. Since AEA and R-methanandamide are structurally similar to arachidonic acid, they may interact with the mAChR in a similar manner to inhibit receptor function.

Inhibition of antagonist and agonist binding to the human brain muscarinic receptor by arachidonic acid

Arachidonic acid (AA) inhibits the binding of [3H]quinclidinyl benzilate ([3H]QNB) to the human brain muscarinic cholinergic receptor (mAChR). AA inhibits at lower concentrations in the absence of glutathione (I50=15 µM) than in the presence of glutathione (I50=42 µM). Inhibition of mAChR binding shows specificity for AA and is reduced with loss of one or more double bonds or with either a decrease or increase in the length of the fatty acid chain. Metabolism of AA by the lipoxygenase, epoxygenase, or fatty acid cyclooxygenase pathways is not required for the inhibitory activity of AA on mAChR binding. Inhibition of [3H]QNB binding by AA is reversible. While decreasing Bmax, AA increased the apparent KD for [3H]QNB and for the more polar antagonist [3H]NMS. In addition, AA inhibits binding of the agonist [3H]oxotremorine-M (I50=60 µM) and is the first mediator of mAChR action to be shown to reversibly inhibit mAChR binding. The feedback inhibition of the mAChR by AA may serve a homeostatic function similar to the reuptake and hydrolysis of acetylcholine following cholinergic nerve transmission.

Previously reported nerve growth factor levels are underestimated due to an incomplete release from receptors and interaction with standard curve media

The 1996 research report by Hoener et al. [M.C. Hoener, E. Hewitt, J. M. Conner, J.W. Costello, S. Varon, Nerve growth factor (NGF) content in adult rat brain tissue is several-fold higher than generally reported and is largely associated with sedimentable fractions, Brain Res. 728 (1996) 47-56.] compares levels of nerve growth factor (NGF) found in rat brain by assaying both supernatant and pellet to previously reported data. However, Hoener et al. miscalculated when converting values previously reported in the literature to units of picogram per milliliter. Regardless of this mistake, the method of tissue extraction does affect the extent of release of NGF, which must be maximized in order to accurately determine NGF levels in the central nervous system. We now report that accurate measurement of NGF levels is not only affected by the incomplete release of NGF from receptors, but also the medium in which the standard curve is run. It is the combination of these two variables that has led to the underestimation of NGF levels in previous research.

Inhibition of ligand binding to g protein-coupled receptors by arachidonic acid

Arachidonic acid (AA), released in response to muscarinic acetylcholine receptor (mAChR) stimulation, previously has been reported to function as a reversible feedback inhibitor of the mAChR. To determine if the effects of AA on binding to the mAChR are subtype specific and whether AA inhibits ligand binding to other G protein-coupled receptors (GPCRs), the effects of AA on ligand binding to the mAChR subtypes (M1, M2, M3, M4, and M5) and to the μ-opioid receptor, β2-adrenergic receptor (β2-AR), 5-hydroxytryptamine receptor (5-HTR), and nicotinic receptors were examined. AA was found to inhibit ligand binding to all mAChR subtypes, to the β2-AR, the 5-HTR, and to the μ-opioid receptor. However, AA does not inhibit ligand binding to the nicotinic receptor, even at high concentrations of AA. Thus, AA inhibits several types of GPCRs, with 50% inhibition occurring at 3–25 µM, whereas the nicotinic receptor, a non-GPCR, remains unaffected. Further research is needed to determine the mechanism by which AA inhibits GPCR function.