Intranasal delivery is a novel non-invasive method of bypassing the blood-brain barrier (BBB) to deliver therapeutic agents to the central nervous system (CNS). Intranasal delivery may be of particular benefit in neurological disorders involving the loss of a specific peptide or protein. The hypothalamic hypocretin neuropeptides (hypocretin 1 and 2) have been linked to regulation of sleep and wakefulness. Deficiency in hypocretin neurotransmission results in narcolepsy in mice, dogs, and humans. As a first step toward assessing the therapeutic potential of intranasal hypocretin 1 replacement for treatment of narcolepsy, we demonstrate direct delivery of intranasal hypocretin 1 from the nose to the brain using gamma counting and autoradiography. In addition, a new method of intranasal delivery to awake mice, allowing rapid repeated dosing over extended periods of time, is reported.

INTRODUCTION

Intranasal Delivery
The blood-brain barrier has prevented the use of many therapeutic agents for treating CNS disorders.¹ Frey has proposed intranasal delivery as an important novel route for bypassing the BBB to deliver therapeutic agents to the brain and spinal cord.² This method allows drugs that do not cross the BBB to be delivered to the CNS within minutes. It also directly targets drugs that do cross the BBB to the CNS, thus eliminating the need for systemic delivery and reducing peripheral side effects. This method works because of the unique connection that the olfactory and trigeminal nerves (involved in sensing odors and chemicals) provide between the brain and the external environment. Delivery occurs by an extracellular route and does not require that the drugs bind to any receptor or undergo axonal transport. A large variety of therapeutic agents have been non-invasively delivered to the CNS directly from the nose, thus bypassing the BBB.²

Since the review published by Frey in 2002, additional studies have confirmed the use of drug delivery to the brain along the intranasal pathway and have demonstrated therapeutic efficacy in animal models of disease. In a transgenic mouse model of Alzheimer's disease, intranasal nerve growth factor has been shown to successfully reduce neurodegeneration.³ Intranasal neurotrophins have also been reported to stimulate neurogenesis in normal adult mice.⁴ In a rat model of stroke, intranasal insulin-like growth factor I, given up to 4 hours after stroke, successfully decreased neurologic damage, suggesting there is a clinically relevant therapeutic window for treatment.⁵ For the treatment of brain cancer, intranasal methotrexate administration to rats was shown to bypass the BBB to target the brain and reduce tumor size, raising the possibility of targeted chemotherapy in humans without side effects.^6,7 In humans, drug delivery to the cerebrospinal fluid after intranasal administration was demonstrated for neuropeptides, including melanocyte stimulating hormone (MSH), insulin, and vasopressin.⁸ Also, acute intranasal insulin is reported to improve memory in patients with Alzheimer's disease, even though there were no changes in blood levels of insulin or glucose.⁹

Hypocretin & Treatment of Narcolepsy
Two groups independently discovered the hypocretin (orexin) peptides (hypocretin 1 and 2; also called orexin A and B) derived from the same precursor peptide, preprohypocretin.^10-13 The role of the individual hypocretin peptides in normal physiology has not been determined, but hypocretin 1 has generally been used in studies because of its greater biological stability and activity. Hypocretin peptides are synthesized exclusively in the lateral hypothalamus, in neurons that project throughout the CNS. Two seven transmembrane G protein-coupled receptors for the hypocretins have been described: hcrtr1 and hcrtr2. In vitro, hypocretin 1 has two to three times greater affinity than hypocretin 2 for the human hcrtr1 receptor. Both peptides have similar affinities for the human hcrtr2 receptor.

Several lines of evidence have pointed to the importance of the hypocretin system in the regulation of wakefulness. Importantly, this neuropeptidergic system is key to the pathophysiology of the common sleep disorder, narcolepsy. Prepro-hypocretin and hcrtr2 gene knockout mice have abnormal behaviour patterns similar to human narcolepsy.^14,15 A canine model of narcolepsy that is inherited in an autosomal recessive fashion is associated with mutations in the hcrtr2 receptor gene.¹⁶ In human narcolepsy, mutations in hypocretin-associated genes are rare, but most patients have hypocretin deficiency in their cerebrospinal fluid.^17,18 In a small post-mortem brain study, brains from narcolepsy patients had undetectable hypothalamic preprohypocretin mRNA by in situ hybridisation.¹⁸ It is believed that in narcolepsy, hypocretin neurons degenerate possibly secondary to an autoimmune insult because narcolepsy is highly associated with the human leukocyte antigen HLA-DQB1*0602.¹³

Intranasal Delivery of Hypocretin 1
Whatever the precise etiology of narcolepsy, replacement of hypocretin peptides appears to be an attractive method of treatment, which unlike currently available stimulant drugs, would be directed at the disease pathology. Intravenous replacement has proved to be ineffective because of poor penetration of these peptides into the brain.¹⁹ The intranasal route may therefore be a useful method for introducing these peptides into the CNS. As a first step toward evaluating the therapeutic potential of intranasal hypocretin 1 as a treatment for narcolepsy, the main objective of this study was to determine if intranasal hypocretin 1 reaches the brain.

MATERIALS & METHODS

Animals
The Institutional Animal Care and Use Committee at HealthPartners Research Foundation approved all experimental procedures. A total of 18 Male Swiss Webster (22-g mean, 0.5-g SE) were used for experiments. Mice were maintained in cages with free access to food and water and kept on a 12-h light-dark schedule.

Intranasal & Intravenous Hypocretin 1 Administration to Anesthetized Mice Hypocretin 1 was administered under anesthesia to all mice except the four that were given awake intranasal delivery described below. Mice were anesthetized with intraperitoneal sodium pentobarbital (Nembutal, 60 mg/kg), and given additional doses throughout the experiment as needed. A heating pad and rectal thermal probe were used to maintain body temperature at 37°C.

Hypocretin 1 was custom 125I-labeled by Amersham Biosciences. A mixture of 125I-hypocretin 1 and unlabeled hypocretin 1 was intranasally delivered and contained an average of 5.5 nmol and 15.6 µCi (n=5). Intranasal hypocretin 1 was administered in eight, 3-µL drops using gel-loading pipette tips, alternating nares every 2 minutes. Intravenous hypocretin 1 (26-µL hypocretin 1 plus 125-µL saline) was administered as a bolus through a tail vein injection over 1 min using a 1-cc syringe with a 30-gauge needle. A mixture of 125I-hypocretin 1 and unlabeled hypocretin 1 was intravenously delivered and contained an average of 5.7 nmol and 16.2 µCi (n=4).

For autoradiographic studies, hypocretin 1 was delivered intranasally at 5.7 nmol and 16.2 µCi (n=3), and intravenously at 5.7 nmol and 16.1 µCi (n=2).

Awake Intranasal Administration of Hypocretin 1
Future studies of intranasal hypocretin 1 as a treatment for narcolepsy will require daily intranasal treatment for several weeks, and repeated anesthetization is not feasible for long-term treatment. Therefore, hypocretin 1 was intranasally delivered to a group of four mice using an awake method. Mice were grabbed by the skin of their necks and held firmly, but gently in the palm of the hand to minimize movement. Drops of 9 µL were administered into alternating nares every 2 min (total 4 drops). Mice were allowed to freely move in a cage between drops. After the last intranasal drop, mice were anesthetized as described previously.

Tissue Collection
Approximately 25 min after the onset of drug delivery, mice were transcardially perfused with 15-mL 0.9% NaCl followed by 115-mL fixative (4% paraformaldehyde in Sorenson's phosphate buffer). Saline and fixative solutions were manually delivered at rate of approximately 5 mL per minute.

Quantitative Measurement of 125I-labeled Hypocretin 1 Distribution
The brain, spinal cord, and peripheral tissues were carefully dissected into individual anatomical areas and weighed, with special attention to avoid cross-contamination (Table 1). Tissue and blood samples were placed in 5-mL Sarstedt tubes for gamma ray counting by a Packard Cobra II Auto-Gamma counter. The concentration of hypocretin 1 in each tissue was calculated using the counts per minute, tissue weight, the hypocretin 1 molecular weight of approximately 3500, and specific activity measurements from standard samples of the hypocretin 1 mixture delivered.

Qualitative Measurement of 125I-labeled Hypocretin 1 Distribution
Brains were sliced into 1-mm coronal sections using a mouse brain matrix. Tissue slices were placed onto glass microscope slides, covered with plastic wrap, and covered with a Super Sensitive (ST) phosphor screen (Packard) for 21 days in an autoradiography cassette. Phosphor screens were developed using a cyclone phosphor scanner (Packard), and data were analyzed with Packard Optiquant software.

Statistical Analysis
All data are expressed as the mean and standard error of the mean (SEM). Comparisons between intranasal and intravenous hypocretin 1 concentrations in each individual tissue were calculated using a student's t-test. A value of P < 0.05 was used to determine statistically significant differences between groups.

RESULTS

Quantitative Distribution of Intranasal Hypocretin 1
Intranasal administration of 5.5 nmol of hypocretin 1 resulted in substantial delivery throughout the brain and spinal cord (Table 1). The highest CNS concentrations of hypocretin 1 following intranasal delivery were found in the trigeminal nerve (1007 nM), followed by olfactory bulbs (324 nM), and anterior olfactory nucleus (126 nM). Concentrations ranged from 14 nM to 61 nM in other regions of the brain (ie, cortex, hippocampus, brain stem) with the highest concentration (61 nM) observed in the hypothalamus (Table 1). In addition to delivery to the brain, intranasal hypocretin 1 also resulted in delivery to the spinal cord, with a decreasing gradient from cervical (96 nM) to lumbar (3 nM) regions. The blood concentration of hypocretin 1 at the time of transcardial perfusion, approximately 25 min after the onset of drug delivery, was 31 nM. Concentrations of hypocretin 1 after intranasal delivery were similar in all peripheral organs (13 nM, muscle; 9 nM, liver; 14 nM, kidney; and 14 nM, lung).

Comparison of Intranasal Versus Intravenous Delivery of Hypocretin 1
The distribution of intravenously administered hypocretin 1 was examined to determine what portion of intranasal delivery to the brain is due to absorption from the nasal cavity, into the blood, and across the BBB into the brain as opposed to direct delivery to the brain via the neural pathways. Intranasal and intravenous doses of hypocretin 1 were similar (5.5 nmol and 15.6 µCi intranasal, and 5.7 nmol and 16.2 µCi intravenous). While the blood concentration from intravenous hypocretin 1 (321 nM) was 10 times higher than that from intranasal hypocretin 1 (31 nM), brain concentrations were approximately half of those achieved with intranasal delivery (Figure 1a). Similar concentrations of hypocretin 1 were seen in all areas of the brain after intravenous administration (~10 nM), with the highest concentration observed in the trigeminal nerve (49 nM) and hypothalamus (15 nM). Concentrations of hypocretin 1 in peripheral organs after intravenous administration were higher than with intranasal administration, especially in the kidney (715 nM versus 14 nM) (Figure 1b). Intravenous administration resulted in similar concentrations of hypocretin 1 across all regions of the spinal cord (17 nM) (Table 1).

Autoradiography of Hypocretin 1 Distribution
The qualitative distribution of hypocretin 1 was examined using autoradiographic analysis to confirm the gamma counting data and to obtain greater detail in the distribution of hypocretin 1 throughout the brain. Autoradiographic images demonstrated that intranasal delivery of hypocretin 1 (5.7 nmol, 16.2µCi) resulted in drug distribution throughout the entire brain, with high concentrations present in areas of the anterior olfactory nucleus (Figure 2a), hypothalamus (Figure 2b), and pontine nuclei (Figure 2c). Autoradiography also confirmed that intranasal hypocretin 1 resulted in greater delivery throughout the brain compared to intravenous hypocretin 1 (Figure 2).

Awake Intranasal Delivery of Hypocretin 1
Intranasal delivery of hypocretin 1 to awake mice (6.9 nmol) resulted in lower brain concentrations than administration to anesthetized mice. Awake mice spent approximately 3 to 4 min on their backs for delivery, while anesthetized mice were on their backs for 25 min. This resulted in less drug reaching the olfactory epithelium as well as less time the drug had contact with the olfactory epithelium. This explains the much lower concentration of hypocretin 1 seen in the olfactory epithelium and olfactory bulb with intranasal delivery to awake mice.

The distribution of hypocretin 1 in the brain was similar with intranasal administration to both anesthetized and awake mice. The highest CNS concentrations of hypocretin 1 following awake intranasal delivery were found in the trigeminal nerve (92 nM), followed by olfactory bulbs (25 nM), and anterior olfactory nucleus (17 nM). Concentrations ranged from 5 nM to 13 nM in other regions of the brain, with the highest concentration (13 nM) again observed in the hypothalamus (Table 2). Awake intranasal hypocretin 1 also resulted in a decreasing gradient in the spinal cord from cervical (23 nM) to lumbar (8 nM) regions. The blood concentration of hypocretin 1 at the time of transcardial perfusion was 140 nM. Similar concentrations of hypocretin 1 after awake intranasal delivery were seen in all peripheral organs (18 nM to 44 nM), with the highest concentration observed in the kidneys.

DISCUSSION

Intranasal delivery is rapidly emerging as an important non-invasive alternative for delivering therapeutic agents to the brain. These results demonstrate that intranasally delivered hypocretin 1 to anesthetized and awake mice directly reaches the brain along the olfactory and trigeminal nerves. Concentrations of hypocretin 1 achieved in the brain after intranasal administration to anesthetized mice (14 to 61 nM) and awake mice (6 to13 nM) are in the physiologic range, and would be expected to produce therapeutic effects as the IC50 of hypocretin 1 for the hcrtr1 receptor is 20 nM.11, 20 In addition to intranasal hypocretin 1 directly targeting the brain, systemic exposure was also reduced, which should decrease possible unwanted side effects. Successful demonstration of intranasal delivery of hypocretin 1 to the brain is the first step toward developing intranasal hypocretin 1 as a treatment for narcolepsy.

The distribution of intranasally delivered hypocretin 1 in the brain was not uniform. Specific regions of the brain, including the hypothalamus, cortex, and brainstem, retained higher concentrations of hypocretin 1 than surrounding tissues after intranasal delivery. This distribution may be explained by the distribution of hypocretin receptors. Hcrtr1 receptor mRNA is most abundant in the ventromedial hypothalamus, but has also been detected in the hippocampus and brainstem.²¹ Hcrtr2 receptor mRNA is expressed in the paraventricular hypothalamic nucleus, the subthalamic and thalamic nuclei, the septum, the cerebral cortex, nucleus accumbens, anterior pretectal nucleus, and several regions in the medulla oblongata. The trigeminal nerves, which contain high concentrations of hypocretin 1, enter the brainstem in close proximity to the cervical spinal cord. This could explain the decreasing concentration gradient of hypocretin 1 from cervical to lumbar spinal cord.

This is the first demonstration of direct intranasal delivery to the brain in awake animals. An intranasal delivery method without the use of anesthesia is critical for future studies testing the therapeutic efficacy of intranasal treatments in animal models of neurological disease, which require repeated dosing over several weeks or months. Successful awake intranasal delivery of hypocretin 1 makes it feasible to test hypocretin 1 in animal models of narcolepsy.

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BIOGRAPHIES

Dr. Leah R. Hanson is currently the Assistant Director of the Alzheimer's Research Center in St. Paul, Minnesota. Dr. Hanson earned a PhD in Neuroscience from the University of Minnesota, Twin Cities studying olfaction and a BS in Biology from Drake University in Des Moines, Iowa. Her current research is focused on the treatment of neurologic disorders using intranasal delivery to bypass the blood-brain-barrier and deliver therapeutic proteins and peptides to the brain. Dr. Hanson co-ordinates all intranasal drug delivery studies at the Alzheimer's Research Center.

Dr. Paula M. Martinez is a Research Associate at the Alzheimer's Research Center. She conducts intranasal drug delivery research assessing radiolabeled distribution, pharmacologic signaling, and behavioral changes. Dr. Martinez earned her MD from the University of Cordoba School of Medicine (Cordoba, Spain) and a BS in Biology from the College of Virgen del Carmen (Cordoba, Spain).

Dr. Emmanuel Mignot, Professor of Psychiatry and Behavioral Sciences and Director of the Center for Narcolepsy, is internationally recognized as having discovered the cause of narcolepsy. He is also known for his discovery of a polymorphism of the "clock" gene that appears to alter individuals' internal biorhythms and for the finding of a gene variant that predisposes to sleep apnea. Dr. Mignot is a former student of the Ecole Normale Superieure (Ulm, Paris, France). He earned his MD and PhD from Paris V and VI University in France. He practiced medicine in France for several years before serving as a visiting scholar at the Stanford Sleep Disorders Clinic and Research Center and later as a visiting assistant professor at Stanford. He joined the Stanford faculty as acting assistant professor of psychiatry and behavioral sciences and was named director of the Stanford Center for Narcolepsy in 1993. Dr. Mignot was named professor of psychiatry and behavioral sciences in 2001. He has published more than 100 original papers, serves on the editorial advisory boards of related journals, is an active member of several professional and governmental organizations, currently chairs the National Center on Sleep Disorders Research Advisory Board of the National Institutes of Health, and received numerous research grants and honors.

Ms. Lyla Kamsheh is currently a second-year medical student at St. George's University in Grenada. Ms. Kamsheh earned her BA in Biology at St. Olaf College in Northfield, Minnesota.

Dr. Shahrad Taheri is lecturer in Medicine and Endocrinology at Bristol University, UK. He studied physiology at King's College, London followed by studies in Human Biology at Oxford University. He then obtained his medical degree from London University in 1994. He became a Wellcome Trust research fellow at the Hammersmith Hospital, Imperial College in 1998 obtaining his PhD investigating the physiological role of hypocretins. He spent 2 years as a research associate at the Howard Hughes Medical Institute, Stanford University studying the role of hypocretins in sleep, the autoimmune basis of narcolepsy, and the interactions between sleep and metabolic hormones.

Dr. William H. Frey, II currently serves as Director of the Alzheimer's Research Center at Regions Hospital in St. Paul, Professor of Pharmaceutics at the University of Minnesota, and consultant to the pharmaceutical and biotechnology industries. His patents, owned by Chiron Corporation and the HealthPartners Research Foundation, target non-invasive delivery of therapeutic and diagnostic agents to the brain and spinal cord for treating neurologic and psychiatric disorders and the use of antioxidants to treat and prevent disease. Dr. Frey has been interviewed on Walter Cronkite's Universe, the Today Show, Good Morning America, 20/20, All Things Considered, and on many other programs in the United States and abroad. Articles examining Dr. Frey's research have appeared in the Wall Street Journal, The New York Times, Forbes, U.S. News and World Report, the New Scientist, and numerous other journals and newspapers. Dr. Frey earned his BA in Chemistry at Washington University and his PhD in Biochemistry from Case Western Reserve University.