Effects of Localized Hydrophilic Mannitol and Hydrophobic Nelfinavir Administration Targeted to Olfactory Epithelium on Brain Distribution

Many nasally applied compounds gain access to the brain and the central nervous system (CNS) with varying degree. Direct nose-to-brain access is believed to be achieved through nervous connections which travel from the CNS across the cribriform plate into the olfactory region of the nasal cavity. However, current delivery strategies are not targeted to preferentially deposit drugs to the olfactory at cribriform. Therefore, we have developed a pressurized olfactory delivery (POD) device which consistently and non-invasively deposited a majority of drug to the olfactory region of the nasal cavity in rats. Using both a hydrophobic drug, mannitol (log P = -3.1), and a hydrophobic drug, nelfinavir (log P = 6.0), and POD device, we compared brain and blood levels after nasal deposition primarily on the olfactory region with POD or nose drops which deposited primarily on the respiratory region in rats. POD administration of mannitol in rats provided a 3.6-fold (p < 0.05) increase in cortex-to-blood ratio, compared to respiratory epithelium deposition with nose drop. Administration of nelfinavir provided a 13.6-fold (p < 0.05) advantage in cortex-to-blood ratio with POD administration, compared to nose drops. These results suggest that increasing the fraction of drug deposited on the olfactory region of the nasal cavity will result in increased direct nose-to-brain transport.     

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.     

The rostral migratory stream plays a key role in intranasal delivery of drugs into the CNS

Background

The blood brain barrier (BBB) is impermeable to most drugs, impeding the establishment of novel neuroprotective therapies and strategies for many neurological diseases. Intranasal administration offers an alternative path for efficient drug delivery into the CNS. So far, the anatomical structures discussed to be involved in the transport of intranasally administered drugs into the CNS include the trigeminal nerve, olfactory nerve and the rostral migratory stream (RMS), but the relative contributions are debated.

Methods and Findings

In the present study we demonstrate that surgical transection, and the resulting structural disruption of the RMS, in mice effectively obstructs the uptake of intranasally administered radioligands into the CNS. Furthermore, using a fluorescent cell tracer, we demonstrate that intranasal administration in mice allows agents to be distributed throughout the entire brain, including olfactory bulb, hippocampus, cortex and cerebellum.

Conclusions

This study provides evidence of the vital role the RMS has in the CNS delivery of intranasally administered agents. The identification of the RMS as the major access path for intranasally administered drugs into the CNS may contribute to the development of treatments that are tailored for efficient transport within this structure. Research into the RMS needs to continue to elucidate its limitations, capabilities, mechanisms of transport and potential hazards before we are able to advance this technique into human research.     

Effect of intranasal arginine vasopressin on human headache

Arginine vasopressin (AVP), a nonapeptide hormone of posterior pituitary, reaches the central nervous system from systemic blood circulation with a difficulty because of the blood–brain barrier (BBB). The interest has been expressed in the use of the nasal route for delivery of AVP to the brain directly, exploiting the olfactory pathway. Our previous study has demonstrated that AVP in the brain rather than the spinal cord and blood circulation plays an important role in rat pain modulation. For understanding the role of AVP on pain modulation in human, the communication tried to investigate the effect of intranasal AVP on human headache. The results showed that (1) AVP concentration in both plasma and cerebrospinal fluid (CSF) increased significantly in headache patients, who related with the headache level; (2) there was a positive relationship between plasma and CSF AVP concentration in headache patients; and (3) intranasal AVP could relieve the human headache in a dose-dependent manner. The data suggested that intranasal AVP, which was delivered to the brain through olfactory region, could treat human headache and AVP might be a potential drug of pain relief by intranasal administration.     

CSF and Blood Oxytocin Concentration Changes following Intranasal Delivery in Macaque

Oxytocin (OT) in the central nervous system (CNS) influences social cognition and behavior, making it a candidate for treating clinical disorders such as schizophrenia and autism. Intranasal administration has been proposed as a possible route of delivery to the CNS for molecules like OT. While intranasal administration of OT influences social cognition and behavior, it is not well established whether this is an effective means for delivering OT to CNS targets. We administered OT or its vehicle (saline) to 15 primates (Macaca mulatta), using either intranasal spray or a nebulizer, and measured OT concentration changes in the cerebral spinal fluid (CSF) and in blood. All subjects received both delivery methods and both drug conditions. Baseline samples of blood and CSF were taken immediately before drug administration. Blood was collected every 10 minutes after administration for 40 minutes and CSF was collected once post-delivery, at the 40 minutes time point. We found that intranasal administration of exogenous OT increased concentrations in both CSF and plasma compared to saline. Both delivery methods resulted in similar elevations of OT concentration in CSF, while the changes in plasma OT concentration were greater after nasal spray compared to nebulizer. In conclusion our study provides evidence that both nebulizer and nasal spray OT administration can elevate CSF OT levels.     

Permeabilization of the Blood-Brain Barrier via Mucosal Engrafting: Implications for Drug Delivery to the Brain

Utilization of neuropharmaceuticals for central nervous system(CNS) disease is highly limited due to the blood-brain barrier(BBB) which restricts molecules larger than 500Da from reaching the CNS. The development of a reliable method to bypass the BBB would represent an enormous advance in neuropharmacology enabling the use of many potential disease modifying therapies. Previous attempts such as transcranial catheter implantation have proven to be temporary and associated with multiple complications. Here we describe a novel method of creating a semipermeable window in the BBB using purely autologous tissues to allow for high molecular weight(HMW) drug delivery to the CNS. This approach is inspired by recent advances in human endoscopic transnasal skull base surgical techniques and involves engrafting semipermeable nasal mucosa within a surgical defect in the BBB. The mucosal graft thereby creates a permanent transmucosal conduit for drugs to access the CNS. The main objective of this study was to develop a murine model of this technique and use it to evaluate transmucosal permeability for the purpose of direct drug delivery to the brain. Using this model we demonstrate that mucosal grafts allow for the transport of molecules up to 500 kDa directly to the brain in both a time and molecular weight dependent fashion. Markers up to 40 kDa were found within the striatum suggesting a potential role for this technique in the treatment of Parkinson’s disease. This proof of principle study demonstrates that mucosal engrafting represents the first permanent and stable method of bypassing the BBB thereby providing a pathway for HMW therapeutics directly into the CNS.     

Improved delivery of broadly neutralizing antibodies by nanocapsules suppresses SHIV infection in the CNS of infant rhesus macaques

Broadly neutralizing antibodies (bNAbs) directed to HIV-1 have shown promise at suppressing viremia in animal models. However, the use of bNAbs for the central nervous system (CNS) infection is confounded by poor penetration of the blood brain barrier (BBB). Typically, antibody concentrations in the CNS are extremely low; with levels in cerebrospinal fluid (CSF) only 0.1% of blood concentrations. Using a novel nanotechnology platform, which we term nanocapsules, we show effective transportation of the human bNAb PGT121 across the BBB in infant rhesus macaques upon systemic administration up to 1.6% of plasma concentration. We demonstrate that a single dose of PGT121 encased in nanocapsules when delivered at 48h post-infection delays early acute infection with SHIV_SF162P3 in infants, with one of four animals demonstrating viral clearance. Importantly, the nanocapsule delivery of PGT121 improves suppression of SHIV infection in the CNS relative to controls.     

Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would. Dual-variable-domain immunoglobulin (DVD-Ig) proteins offer a bispecific format where monoclonal antibody-like bivalency to both the BBB receptor and the therapeutic target is preserved, enabling independent engineering of binding affinity, potency, valency, epitope and conformation, essential for successful generation of clinical candidates for CNS applications with desired drug-like properties. Each of these parameters can affect the binding and transcytosis ability mediated by different receptors on the brain endothelium differentially, allowing exploration of diverse properties. Here, we describe generation and characterization of several different DVD-Ig proteins, specific for four different CNS targets, capable of crossing the BBB through transcytosis mediated by the transferrin receptor 1 (TfR1). After systemic administration of each DVD-Ig, we used two independent methods in parallel to observe specific uptake into the brain. An electrochemiluminescent-based sensitive quantitative assay and a semi-quantitative immunohistochemistry technique were used for brain concentration determination and biodistribution/localization in brain, respectively. Significantly enhanced brain uptake and retention was observed for all TfR1 DVD-Ig proteins regardless of the CNS target or the systemic administration route selected.     

Quetiapine Nanoemulsion for Intranasal Drug Delivery: Evaluation of Brain-Targeting Efficiency

To evaluate the possibility of improved drug delivery of quetiapine fumarate (QTP), a nanoemulsion system was developed for intranasal delivery. Effects of different HLBs of Emalex LWIS 10, PEG 400 and Transcutol P, as co-surfactants, were studied on isotropic region of pseudoternary-phase diagrams of nanoemulsion system composed of capmul MCM (CPM) as oil phase, Tween 80 as surfactant and water. Phase behaviour, globule size, transmission electron microscope (TEM) photographs and brain-targeting efficiency of quetiapine nanoemulsion were investigated. In vitro dissolution study of optimised nanoemulsion formulation, with mean diameter 144?±?0.5 nm, showed more than twofold increase in drug release as compared with pure drug. According to results of in vivo tissue distribution study in Wistar rats, intranasal administration of QTP-loaded nanoemulsion had shorter T _max compared with that of intravenous administration. Higher drug transport efficiency (DTE%) and direct nose-to-brain drug transport (DTP%) was achieved by nanoemulsion. The nanoemulsion system may be a promising strategy for brain-targeted delivery of QTP.     

Targeted delivery of antibodies through the blood-brain barrier by MRI-guided focused ultrasound

The blood-brain barrier (BBB) is a persistent obstacle for the local delivery of macromolecular therapeutic agents to the central nervous system (CNS). Many drugs that show potential for treating CNS diseases cannot cross the BBB and there is a need for a non-invasive targeted drug delivery method that allows local therapy of the CNS using larger molecules. We developed a non-invasive technique that allows the image-guided delivery of antibody across the BBB into the murine CNS. Here, we demonstrate that subsequent to MRI-targeted focused ultrasound induced disruption of BBB, intravenously administered dopamine D(4) receptor-targeting antibody crossed the BBB and recognized its antigens. Using MRI, we were able to monitor the extent of BBB disruption. This novel technology should be useful in delivering macromolecular therapeutic or diagnostic agents to the CNS for the treatment of various CNS disorders.     

Nose-To-Brain Delivery of PLGA-Diazepam Nanoparticles

The objective of the present investigation was to optimize diazepam (Dzp)-loaded poly(lactic-co-glycolic acid) nanoparticles (NP) to achieve delivery in the brain through intranasal administration. Dzp nanoparticles (DNP) were formulated by nanoprecipitation and optimized using Box-Behnken design. The influence of various independent process variables (polymer, surfactant, aqueous to organic (w/o) phase ratio, and drug) on resulting properties of DNP (z-average and drug entrapment) was investigated. Developed DNP showed z-average 148–337 d.nm, polydispersity index 0.04–0.45, drug entrapment 69–92%, and zeta potential in the range of −15 to −29.24 mV. Optimized DNP were further analyzed by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), ex-vivo drug release, and in-vitro cytotoxicity. Ex-vivo drug release study via sheep nasal mucosa from DNP showed a controlled release of 64.4% for 24 h. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay performed on Vero cell line showed less toxicity for DNP as compared to Dzp suspension (DS). Gamma scintigraphy and biodistribution study of DNP and DS was performed on Sprague-Dawley rats using technetium-99m-labeled (^99mTc) Dzp formulations to investigate the nose-to-brain drug delivery pathway. Brain/blood uptake ratios, drug targeting efficiency, and direct nose-to-brain transport were found to be 1.23–1.45, 258, and 61% for ^99mTc-DNP (i.n) compared to ^99mTc-DS (i.n) (0.38–1.06, 125, and 1%). Scintigraphy images showed uptake of Dzp from nose-to-brain, and this observation was in agreement with the biodistribution results. These results suggest that the developed poly(D,L-lactide-co-glycolide) (PLGA) NP could serve as a potential carrier of Dzp for nose-to-brain delivery in outpatient management of status epilepticus.KEY WORDS: controlled release, nanoparticles, process optimization, scintigraphy     

A New Brain Drug Delivery Strategy: Focused Ultrasound-Enhanced Intranasal Drug Delivery

Central nervous system (CNS) diseases are difficult to treat because of the blood-brain barrier (BBB), which prevents most drugs from entering into the brain. Intranasal (IN) administration is a promising approach for drug delivery to the brain, bypassing the BBB; however, its application has been restricted to particularly potent substances and it does not offer localized delivery to specific brain sites. Focused ultrasound (FUS) in combination with microbubbles can deliver drugs to the brain at targeted locations. The present study proposed to combine these two different platform techniques (FUS+IN) for enhancing the delivery efficiency of intranasally administered drugs at a targeted location. After IN administration of 40 kDa fluorescently-labeled dextran as the model drug, FUS targeted at one region within the caudate putamen of mouse brains was applied in the presence of systemically administered microbubbles. To compare with the conventional FUS technique, in which intravenous (IV) drug injection is employed, FUS was also applied after IV injection of the same amount of dextran in another group of mice. Dextran delivery outcomes were evaluated using fluorescence imaging of brain slices. The results showed that FUS+IN enhanced drug delivery within the targeted region compared with that achieved by IN only. Despite the fact that the IN route has limited drug absorption across the nasal mucosa, the delivery efficiency of FUS+IN was not significantly different from that of FUS+IV. As a new drug delivery platform, the FUS+IN technique is potentially useful for treating CNS diseases.     

Intranasal Delivery of Recombinant NT4-NAP/AAV Exerts Potential Antidepressant Effect

The present study was designed to construct a recombinant adeno-associated virus (rAAV) which can express NAP in the brain and examine whether this virus can produce antidepressant effects on C57 BL/6 mice that had been subjected to open field test and forced swimming test, via nose-to-brain pathway. When the recombinant plasmid pGEM-T Easy/NT4-NAP was digested by EcoRI, 297 bp fragments can be obtained and NT4-NAP sequence was consistent with the designed sequence confirmed by DNA sequencing. When the recombinant plasmid pSSCMV/NT4-NAP was digested by EcoRI, 297 bp fragments is visible. Immunohistochemical staining of fibroblasts revealed that expression of NAP was detected in NT4-NAP/AAV group. Intranasal delivery of NT4-NAP/AAV significantly reduced immobility time when the FST was performed after 1 day from the last administration. The effects observed in the FST could not be attributed to non-specific increases in activity since intranasal delivery of NT4-NAP/AAV did not alter the behavior of the mice during the open field test. The results indicated that a recombinant AAV vector which could express NAP in cells was successfully constructed and NAP may be a potential target for therapeutic action of antidepressant treatment.     

Targeted Delivery of GDNF through the Blood–Brain Barrier by MRI-Guided Focused Ultrasound

Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), are promising therapeutic agents for neurodegenerative diseases. However, the application of GDNF to treat these diseases effectively is limited because the blood–brain barrier (BBB) prevents the local delivery of macromolecular therapeutic agents from entering the central nervous system (CNS). Focused ultrasound combined with microbubbles (MBs) using appropriate parameters has been previously demonstrated to be able to open the BBB locally and noninvasively. This study investigated the targeted delivery of GDNF MBs through the BBB by magnetic resonance imaging (MRI)-guided focused ultrasound. Evans Blue extravasation and histological examination were used to determine the optimum focused ultrasound parameters. Enzyme-linked immunosorbent assay was performed to verify the effects of GDNF bound on MBs using a biotin–avidin bridging chemistry method to promote GDNF delivery into the brain. The results showed that GDNF can be delivered locally and noninvasively into the CNS through the BBB using MRI-guided focused ultrasound combined with MBs under optimum parameters. MBs that bind GDNF combined with MRI-guided focused ultrasound may be an effective way of delivering neurotrophic factors directly into the CNS. The method described herein provides a potential means of treating patients with CNS diseases.     

Pulmonary gene delivery using polymeric nonviral vectors

Pulmonary delivery provides an easy and well tolerated means of access for the administration of biomacromolecules to the pulmonary epithelium and could therefore be an attractive approach for local and systemic therapies. A growing number of reports, which are summarized in this review, mirror the viability of pulmonary gene delivery. Special attention has been paid to the biological barriers in the lung that must be overcome for successful delivery, and which can be divided into anatomic, physical, immunologic, and metabolic barriers. In light of these barriers, successful nonviral polymer-based formulations of therapeutic genes are presented depending on the chemical nature of the polymer. In addition to polyethyleneimine-based nonviral vectors, which have been most intensively studied for pulmonary gene delivery in the past, other polymeric, dendritic, and targeted materials are also described here, including novel and biodegradable polymers. As new materials need in vitro or ex vivo testing before in vivo application, sophisticated models for all three approaches have been illustrated. Although pulmonary siRNA delivery enjoys popularity in clinical trials, pulmonary gene delivery has so far not been translated into clinical applications. With this review, potential hurdles are demonstrated, but novel approaches that may lead to optimized systems are described as well.     

A novel LRP1-binding peptide L57 that crosses the blood brain barrier

The blood-brain barrier (BBB) is a major obstacle to drug delivery into the central nervous system (CNS), in particular for macromolecules such as peptides and proteins. However, certain macromolecules can reach the CNS via a receptor-mediated transcytosis (RMT) pathway, and low-density lipoprotein receptor-related protein 1 (LRP1) is one of the promising receptors for RMT. An LRP1 ligand peptide, Angiopep-2, was reported to pass through the BBB and deliver covalently conjugated drugs into the CNS. While conjugation of LRP1 ligands with drugs would be an effective approach for drug delivery to the CNS, no other reliable LRP1 ligands have been reported to date. In this study, we aimed to identify novel LRP1 ligands to further investigate LRP1-mediated RMT. Using phage display technology, we obtained a novel peptide, L57 (TWPKHFDKHTFYSILKLGKH-OH), with an EC₅₀ value of 45 nM for binding to cluster 4 (Ser3332–Asp3779) of LRP1. L57 was stable in mouse plasma for up to 20 min. In situ brain perfusion assay in mice revealed the significantly high BBB permeability of L57. In conclusion, we discovered L57, the first artificial LRP1-binding peptide with BBB permeability. Our findings will contribute to the development of RMT-based drugs for the treatment of CNS diseases.     

WR-2721 entry into the brain across a modified blood-brain barrier

Radioprotection of the CNS by WR-2721 has not been possible because of its inability to cross the blood-brain barrier (BBB) and so gain access to the neural tissue. Modification of the BBB using hypertonic arabinose (1.8 m), injected via the internal carotid artery (ica), permitted entry of ip-injected [14C]WR-2721 into the ipsilateral cerebral hemisphere. The BBB-modified hemisphere had a 5.34-fold increased uptake compared to nonmodified controls. Delivery as a bolus via the ica further enhanced uptake after BBB opening; WR-2721 was 3.73 times greater than by ip injection. A 20-fold increase of WR-2721 brain uptake has been calculated for ica administration with the BBB opened as compared to the ip route without BBB modification. Toxicity of ip-administered WR-2721 with the BBB open was only 1.4 times greater than non-modified controls and 1.96 times more toxic when delivered via the ica. These data demonstrate significant uptake of WR-2721 into the CNS, a previously unprotected organ, and provide a model for future radioprotective studies.     

Pharmacokinetic behavior in plasma, cerebrospinal fluid and cerebral cortex after intranasal administration of hydrochloride meptazinol

The aim of this paper is to investigate the pharmacokinetic behavior of hydrochloride meptazinol (MEP) in plasma, cerebrospinal fluid (CSF) and cerebral cortex after intranasal administration (8 mg/kg) in male Sprague-Dawley rats. The pharmacokinetic study of intravenous administration (8 mg/kg) was also performed in rats. CSF and cerebral cortex samples were collected by serial CSF sampling and intracerebral microdialysis, respectively. The concentration of MEP in the biological samples was measured by high performance liquid chromatography (HPLC). It was determined that the absorption of MEP from the nasal cavity to systemic circulation was rapid and complete. The concentration-time profile showed a prolonged duration of MEP concentration in CSF and cortex following intranasal administration. The ratios of AUC values of intranasal to intravenous administrations were 0.96, 1.07 and 1.81 in plasma, CSF and cortex dialysate, respectively. In conclusion, intranasal administration of MEP is a promising alternative to traditional administration modes. Olfactory mucosa did not present intranasal MEP another pathway, in addition to systemic absorption, for transport to the brain.     

A comparative analysis of the distribution of glyprolines after their administration by different ways

The distribution of the glyprolines, Pro-Gly-Pro and Thr-Lys-Pro-Arg-Pro-Gly-Pro (Selanc), was analyzed and compared in tissues of rat organs after different ways of their administration using the peptides uniformly labeled with tritium. Comparative data on changes of concentrations of the peptides in the rat organs after their intraperitoneal, intranasal, intragastric, and intravenous administration are given. The intranasal administration of both peptides was shown to be optimal for delivery of glyprolines molecules in the CNS. A high affinity of the studied glyprolines for gastric tissues was found for all the ways of their administration. We suggest that high efficacy of action of glyprolines on homeostasis of the gastric mucosa was partially provided by accumulation of these peptides (to high concentrations) in gastric tissues.