Mechanisms of Intranasal Deferoxamine in Neurodegenerative and Neurovascular Disease.

Identifying disease-modifying therapies for neurological diseases remains one of the greatest gaps in modern medicine. Herein, we present the rationale for intranasal (IN) delivery of deferoxamine (DFO), a high-affinity iron chelator, as a treatment for neurodegenerative and neurovascular disease with a focus on its novel mechanisms. Brain iron dyshomeostasis with iron accumulation is a known feature of brain aging and is implicated in the pathogenesis of a number of neurological diseases. A substantial body of preclinical evidence and early clinical data has demonstrated that IN DFO and other iron chelators have strong disease-modifying impacts in Alzheimer's disease (AD), Parkinson's disease (PD), ischemic stroke, and intracranial hemorrhage (ICH). Acting by the disease-nonspecific pathway of iron chelation, DFO targets each of these complex diseases via multifactorial mechanisms. Accumulating lines of evidence suggest further mechanisms by which IN DFO may also be beneficial in cognitive aging, multiple sclerosis, traumatic brain injury, other neurodegenerative diseases, and vascular dementia. Considering its known safety profile, targeted delivery method, robust preclinical efficacy, multiple mechanisms, and potential applicability across many neurological diseases, the case for further development of IN DFO is considerable.

Intranasal deferoxamine can improve memory in healthy C57 mice, suggesting a partially non-disease-specific pathway of functional neurologic improvement.

INTRODUCTION: Intranasal deferoxamine (IN DFO) has been shown to decrease memory loss and have beneficial impacts across several models of neurologic disease and injury, including rodent models of Alzheimer's and Parkinson's disease. METHODS: In order to assess the mechanism of DFO, determine its ability to improve memory from baseline in the absence of a diseased state, and assess targeting ability of intranasal delivery, we treated healthy mice with IN DFO (2.4 mg) or intraperitoneal (IP) DFO and compared behavioral and biochemical changes with saline-treated controls. Mice were treated 5 days/week for 4 weeks and subjected to behavioral tests 30 min after dosing. RESULTS: We found that IN DFO, but not IP DFO, significantly enhanced working memory in the radial arm water maze, suggesting that IN administration is more efficacious as a targeted delivery route to the brain. Moreover, the ability of DFO to improve memory from baseline in healthy mice suggests a non-disease-specific mechanism of memory improvement. IN DFO treatment was accompanied by decreased GSK-3ß activity and increased HIF-1α activity. CONCLUSIONS: These pathways are suspected in DFO's ability to improve memory and perhaps represent a component of the common mechanism through which DFO enacts beneficial change in models of neurologic disease and injury.

Intranasal delivery of low-dose insulin ameliorates motor dysfunction and dopaminergic cell death in a 6-OHDA rat model of Parkinson's Disease.

Emerging evidence continues to demonstrate that disrupted insulin signaling and altered energy metabolism may play a key role underpinning pathology in neurodegenerative conditions. Intranasally administered insulin has already shown promise as a memory-enhancing therapy in patients with Alzheimer's and animal models of the disease. Intranasal drug delivery allows for direct targeting of insulin to the brain, bypassing the blood brain barrier and minimizing systemic adverse effects. In this study, we sought to expand upon previous results that show intranasal insulin may also have promise as a Parkinson's therapy. We treated 6-OHDA parkinsonian rats with a low dose (3 IU/day) of insulin and assessed apomorphine induced rotational turns, motor deficits via a horizontal ladder test, and dopaminergic cell survival via stereological counting. We found that insulin therapy substantially reduced motor dysfunction and dopaminergic cell death induced by unilateral injection of 6-OHDA. These results confirm insulin's efficacy within this model, and do so over a longer period after model induction which more closely resembles Parkinson's disease. This study also employed a lower dose than previous studies and utilizes a delivery device, which could lead to an easier transition into human clinical trials as a therapeutic for Parkinson's disease.

Quantifying Intranasally Administered Deferoxamine in Rat Brain Tissue with Mass Spectrometry.

Deferoxamine, a metal chelator, has been shown to be neuroprotective in animal models of ischemic stroke, traumatic brain injury and both subarachnoid and intracerebral hemorrhage. Intranasal deferoxamine (IN DFO) has also shown promise as a potential treatment for multiple neurodegenerative diseases, including Parkinson's and Alzheimer's. However, there have been no attempts to thoroughly understand the dynamics and pharmacokinetics of IN DFO. We developed a new high-performance liquid-chromatography electrospray-tandem mass spectrometry (HPLC/ESI-MS2) method to quantify the combined total levels of DFO, ferrioxamine (FO; DFO bound to iron), and aluminoxamine (AO; aluminum-bound DFO) in brain tissue using a custom-synthesized deuterated analogue (DFO-d7, Medical Isotopes Inc., Pelham NH) as an internal standard. We applied our method toward understanding the pharmacokinetics of IN DFO delivery to the brain and blood of rats from 15 min to 4 h after delivery. We found that IN delivery successfully targets DFO to the brain to achieve concentrations of 0.5-15 µM in various brain regions within 15 min, and decreasing though still detectable after 4 h. Systemic exposure was minimized as assessed by concentration in blood serum. Serum concentrations were 0.02 µM at 15 min and no more than 0.1 µM at later time points. Compared to blood serum, brain region-specific drug exposure (as measured by area under the curve) ranged from slightly under 10 times exposure in the hippocampus to almost 200 times exposure in the olfactory bulb with IN DFO delivery. These findings represent a major step toward future method development, pharmacokinetic studies, and clinical trials for this promising therapeutic.

Intranasal Coadministration of a Diazepam Prodrug with a Converting Enzyme Results in Rapid Absorption of Diazepam in Rats.

Intranasal administration is an attractive route for systemic delivery of small, lipophilic drugs because they are rapidly absorbed through the nasal mucosa into systemic circulation. However, the low solubility of lipophilic drugs often precludes aqueous nasal spray formulations. A unique approach to circumvent solubility issues involves coadministration of a hydrophilic prodrug with an exogenous converting enzyme. This strategy not only addresses poor solubility but also leads to an increase in the chemical activity gradient driving drug absorption. Herein, we report plasma and brain concentrations in rats following coadministration of a hydrophilic diazepam prodrug, avizafone, with the converting enzyme human aminopeptidase B Single doses of avizafone equivalent to diazepam at 0.500, 1.00, and 1.50 mg/kg were administered intranasally, resulting in 77.8% ± 6.0%, 112% ± 10%, and 114% ± 7% bioavailability; maximum plasma concentrations 71.5 ± 9.3, 388 ± 31, and 355 ± 187 ng/ml; and times to peak plasma concentration 5, 8, and 5 minutes for each dose level, respectively. Both diazepam and a transient intermediate were absorbed. Enzyme kinetics incorporated into a physiologically based pharmacokinetic model enabled estimation of the first-order absorption rate constants: 0.0689 ± 0.0080 minutes-1 for diazepam and 0.122 ± 0.022 minutes-1 for the intermediate. Our results demonstrate that diazepam, which is practically insoluble, can be delivered intranasally with rapid and complete absorption by coadministering avizafone with aminopeptidase B. Furthermore, even faster rates of absorption might be attained simply by increasing the enzyme concentration, potentially supplanting intravenous diazepam or lorazepam or intramuscular midazolam in the treatment of seizure emergencies.

Rescue therapies for seizure emergencies: New modes of administration.

A subgroup of patients with drug-resistant epilepsy have seizure clusters, which are a part of the continuum of seizure emergencies that includes prolonged episodes and status epilepticus. When the patient or caregiver can identify the beginning of a cluster, the condition is amenable to certain treatments, an approach known as rescue therapy. Intravenous drug administration offers the fastest onset of action, but this route is usually not an option because most seizure clusters occur outside of a medical facility. Alternate routes of administration have been used or are proposed including rectal, buccal, intrapulmonary, subcutaneous, intramuscular, and intranasal. The objective of this narrative review is to describe the (1) anatomical, physiologic, and drug physicochemical properties that need to be considered when developing therapies for seizure emergencies and (2) products currently in development. New therapies must consider parameters of Fick's law such as absorptive surface area, blood flow, membrane thickness, and lipid solubility, because these factors affect both rate and extend of absorption. For example, the lung has a 50 000-fold greater absorptive surface area than that associated with a subcutaneous injection. Lipid solubility is a physicochemical property that influences the absorption rate of small molecule drugs. Among drugs currently used or under development for rescue therapy, allopregnanolone has the greatest lipid solubility at physiologic pH, followed by propofol, midazolam, diazepam, lorazepam, alprazolam, and brivaracetam. However, greater lipid solubility correlates with lower water solubility, complicating formulation of rescue therapies. One approach to overcoming poor aqueous solubility involves the use of a water-soluble prodrug coadministered with a converting enzyme, which is being explored for the intranasal delivery of diazepam. With advances in seizure prediction technology and the development of drug delivery systems that provide rapid onset of effect, rescue therapies may prevent the occurrence of seizures, thus greatly improving the management of epilepsy.

Food consumption and activity levels increase in rats following intranasal Hypocretin-1.

Hypocretin-1 (HC, orexin-A) is a neuropeptide involved in regulating physiological functions of sleep, appetite and arousal, and it has been shown that intranasal (IN) administration can target HC to the brain. Recent clinical studies have shown that IN HC has functional effects in human clinical trials. In this study, we use rats to determine whether IN HC has an immediate effect on food consumption and locomotor activity, whether distribution in the brain after IN delivery is dose-dependent, and whether MAPK and PDK1 are affected after IN delivery. Food intake and wheel-running activity were quantified for 24h after IN delivery. Biodistribution was determined 30min after IN delivery of both a high and low dose of 125I-radiolabelled HC throughout the brain and other bodily tissues, while Western blots were used to quantify changes in cell signaling pathways (MAPK and PDK1) in the brain. Intranasal HC significantly increased food intake and wheel activity within 4h after delivery, but balanced out over the course of 24h. The distribution studies showed dose-dependent delivery in the CNS and peripheral tissues, while PDK1 was significantly increased in the brain 30min after IN delivery of HC. This study adds to the growing body of evidence that IN administration of HC is a promising strategy for treatment of HC related behaviors.

Intranasal deferoxamine engages multiple pathways to decrease memory loss in the APP/PS1 model of amyloid accumulation.

In addition to the hallmark accumulation of amyloid and hyper-phosphorylation of tau, brain changes in Alzheimer's disease are multifactorial including inflammation, oxidative stress, and metal dysregulation. Metal chelators have been explored as a less well known approach to treatment. One chelator currently being developed is deferoxamine (DFO), administered via the intranasal (IN) route. In the current study, APP/PS1 amyloid mice were treated with a chronic, low dose of IN DFO, subjected to a rigorous battery of behavior tests, and the mechanism of action was examined. Mice were treated 3x/week with 0.24 C IN DFO for 18 weeks from 36 to 54 weeks of age, 4 weeks of behavior tests were performed that included both working and reference memory, anxiolytic and motor behaviors, and finally brain tissues were analyzed for amyloid, protein oxidation, and other proteins affected by DFO. We found that IN DFO treatment significantly decreased loss of both reference and working memory in the Morris and radial arm water mazes (p < 0.05), and also decreased soluble Aß40 and Aß42 in cortex and hippocampus (p < 0.05). Further, IN DFO decreased activity of GSK3ß, and led to decreases in oxidative stress (p < 0.05). These data demonstrate that low doses of IN DFO can modify several targets along the multiple pathways implicated in the neuropathology of Alzheimer's, making it an attractive candidate for the treatment of this heterogeneous disease.

Intranasal administration of CNS therapeutics to awake mice.

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials. Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models and deferoxamine in Alzheimer's mouse models. The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

Intranasal delivery of growth differentiation factor 5 to the central nervous system.

CONTEXT: Growth differentiation factor 5 (GDF5), in addition to its role in bone and joint development, protects dopaminergic (DA) neurons from degeneration, and is a potential therapeutic agent for Parkinson's disease. Its large size and insolubility at physiologic pH are obstacles for drug administration to the central nervous system (CNS) in humans. OBJECTIVE: In this study, formulations to deliver GDF5 to the brain using intranasal (IN) administration were developed. MATERIALS AND METHODS: IN administration of GDF5 in acidic buffer, 20 mM sodium acetate (NaAc) at pH 4.25, was performed in rats. Also, a lipid microemulsion (LME) comprised of olive oil and phosphatidylserine (PS) was used to formulate GDF5 at neutral pH for IN administration. Tissue concentrations of GDF5 were determined by both gamma counting and enzyme-linked immunosorbent assay (ELISA). RESULTS: IN administration of GDF5 in acidic buffers bypassed the blood-brain barrier (BBB), resulting in delivery to the brain with limited systemic exposure. IN administration of GDF5-LME increased drug targeting to the midbrain eightfold when compared to IN administration of GDF5 in acidic buffer. DISCUSSION AND CONCLUSION: This study is the first to show that GDF5 can be formulated at neutral pH and can be directly delivered to the CNS via IN administration, with biologically relevant concentrations in the midbrain where it may be used to treat Parkinson's disease.

Intranasal delivery of deferoxamine reduces spatial memory loss in APP/PS1 mice.

Intranasal administration, which bypasses the blood-brain barrier and minimizes systemic exposure, is a non-invasive alternative for targeted drug delivery to the brain. While identification of metal dysregulation in Alzheimer's brain has led to the development of therapeutic metal-binding agents, targeting to the brain has remained an issue. The purpose of this study was to both determine concentrations of deferoxamine (DFO), a high-affinity iron chelator, reaching the brains of mice after intranasal administration and to determine its efficacy in a mouse model of spatial memory loss. Intranasal administration of DFO (2.4 mg) labeled with (59)Fe (75 µCi) to C57 mice resulted in micromolar concentrations at 30 min within brain parenchyma. After 3 months of intranasal DFO treatment, 2.4 mg three times per week, 48-week-old APP/PS1 mice had significantly reduced escape latencies in Morris water maze compared to vehicle-treated mice. This is the first report that intranasal DFO improves spatial memory in a mouse model of Alzheimer's disease and demonstrates that intranasal DFO reaches the brain in therapeutic doses.

Isolation and biological activity of the multi-component sea lamprey migratory pheromone.

Migratory adult sea lampreys locate spawning streams by using a pheromone released by stream-resident conspecific larvae. It was recently reported that this pheromone is comprised of a mixture of three sulfated steroids: petromyzonamine disulfate (PADS), petromyzosterol disulfate (PSDS), and petromyzonol sulfate (PS). This manuscript reports in-depth details of pheromone isolation and provides new information on the olfactory potency of PADS and PSDS and the behavioral activity of synthesized PADS. Isolation was accomplished using bioassay-guided fractionation which included liquid chromatography-mass spectrometry, electro-olfactogram recording (EOG), and behavioral assays. Both highly purified and synthesized PADS stimulated the olfactory system of adult lamprey and were attractive at concentrations of 10(-13) M. PSDS also had olfactory activity at 10(-13) M. Cross-adaptation studies with EOG recording demonstrated that PADS, PSDS, and PS are detected by independent olfactory receptor sites. Finally, the mixture of all three components was as attractive as larval water to adult sea lampreys in laboratory mazes. It is believed that these steroids are the principal components of the pheromone.

A sterol-like odorant in the urine of Mozambique tilapia males likely signals social dominance to females.

Many species of freshwater fish with relatively simple mating strategies release hormonally derived sex pheromones in urine. However, it is not known whether species with more complex reproductive strategies use specialized urinary chemical signals. We addressed this by using the Mozambique tilapia (Oreochromis mossambicus Peters 1852), a lek-breeding species in which males establish dominance hierarchies and visiting females mate preferentially with territorial/dominant males. We measured urination frequency of territorial males in social isolation and in the presence of females that were either ready to spawn or had finished spawning. In groups of fish, we monitored the volume of urine stored in subordinate and dominant males to determine if urine volume and olfactory potency (by recording electro-olfactograms, EOG, in females) are related to the male's social rank. Dominant, territorial males stored more urine than subordinates and released it in short pulses, the frequency of which increased in the presence of females ready to spawn but not in the presence of post-spawn females. Urine from subordinate and dominant males was fractionated by liquid chromatography and fractions tested for olfactory potency by using the EOG, with the most potent fraction analyzed by mass spectrometry (MS). The olfactory system of females was sensitive to a urinary compound that was more abundant in the urine of dominant males than in that of subordinates. MS analysis suggested the compound is a sulfated aminosterol-like compound with a formula of C29H40N2O10S. Therefore, we suggest that dominant/territorial tilapia males dramatically increase urination frequency in the presence of females ready to spawn and that the urinary odorant acts as a pheromonal signal of dominance, thereby influencing female spawning.

Details of the structure determination of the sulfated steroids PSDS and PADS: new components of the sea lamprey (petromyzon marinus) migratory pheromone.

The discovery of two new components of the migratory pheromone used by sea lamprey to guide adults to spawning grounds was recently reported. These hold promise for use in a pheromone-based control program for this species, an invasive pest in the Great Lakes. Details of the structure determination of these steroidal bis-sulfates [petromyzosterol disulfate (PSDS, 2) and petromyzonamine disulfate (PADS, 3)] are described here. Pattern matching of 1H NMR data was particularly valuable. This involved comparison of spectra of the natural samples of 2 and 3 with those of appropriate steroidal analogues [e.g., petromyzonol sulfate (PS, 1, a previously known sea lamprey bile acid derivative that is a third component of the migratory pheromone), cholesterol sulfate (6), and squalamine (8)] and model compounds containing the unprecedented aminolactam substructure present in 3. The logic underlying the iterative analyses used is presented.

Biologically relevant concentrations of petromyzonol sulfate, a component of the sea lamprey migratory pheromone, measured in stream water.

Adult sea lampreys locate spawning streams in the Great Lakes by using a migratory pheromone that is released by stream-resident larval conspecifics. Behavioral, electrophysiological, and biochemical analyses of larval release water have suggested that this pheromone is composed of several components, one of which is petromyzonol sulfate (PS), a known lamprey-specific bile acid. Its precursor, allocholic acid (ACA), has also been implicated. In this study, we employed high-performance liquid chromatography and mass spectrometry to look for both bile acids in various stream waters, thereby testing whether they might have a role in natural pheromone function. Although PS was measured at picomolar concentrations in streams known to contain larval lampreys and attract migratory adults, ACA was not. Neither compound was measured in streams lacking larvae. This finding indicates that PS is a component of the natural pheromone, and it suggests that ACA has little relevance.

Mixture of new sulfated steroids functions as a migratory pheromone in the sea lamprey.

The sea lamprey is an ancient, parasitic fish that invaded the Great Lakes a century ago, where it triggered the collapse of many fisheries. Like many fishes, this species relies on chemical cues to mediate key aspects of its life, including migration and reproduction. Here we report the discovery of a multicomponent steroidal pheromone that is released by stream-dwelling larval lamprey and guides adults to spawning streams. We isolated three compounds with pheromonal activity (in submilligram quantities from 8,000 l of larval holding water) and deduced their structures. The most important compound contains an unprecedented 1-(3-aminopropyl)pyrrolidin-2-one subunit and is related to squalamine, an antibiotic produced by sharks. We verified its structure by chemical synthesis; it attracts adult lamprey at very low (subpicomolar) concentrations. The second component is another new sulfated steroid and the third is petromyzonol sulfate, a known lamprey-specific bile acid derivative. This mixture is the first migratory pheromone identified in a vertebrate and is being investigated for use in lamprey control.

Evidence that petromyzontid lampreys employ a common migratory pheromone that is partially comprised of bile acids.

This study examined whether the larval pheromone employed by adult sea lamprey (Petromyzon marinus) to locate spawning streams and known to be at least partially comprised of bile acids is also employed by other lamprey species. Both production and release of lamprey-specific bile acids, and sensitivity to them were examined in a wide variety of species. High pressure liquid chromatography and electrospray ionization/mass spectrometry (ESI-MS) found gallbladders from 10 species of European and North American lamprey to contain large quantities of petromyzonol sulfate (PS) together with much smaller quantities of allocholic acid (ACA) and petromyzonol (P). Evaluation of holding waters from three of these species using ESI-MS found all to contain large quantities of PS and lesser quantities of ACA in similar ratios. Electro-olfactogram recording from the olfactory systems of three parasitic lamprey species found all to detect PS and ACA with high sensitivity. Behavioral studies using migratory adult sea lamprey found them to be attracted to the odors of heterospecific larvae as well as conspecific larvae, both of which contained similar amounts of PS and ACA. Finally, adult silver lampreys (Ichthyomyzon unicuspis) were also found to be attracted to the odor of larval sea lamprey. Together, these results demonstrate that PS and ACA are commonly produced and released by larval petromyzontid lampreys and likely used as part of a common evolutionarily conserved pheromone. This scenario is reasonable because lampreys share similar larval and spawning habitat requirements, and their larvae derive no apparent benefit from producing compounds that serve as an attractant for adults.