Basic NeuroscienceRapid intranasal delivery of chloramphenicol acetyltransferase in the active form to different brain regions as a model for enzyme therapy in the CNS
Graphical abstract
Introduction
A major obstacle in the treatment of neurotoxicity caused by chemical threat agents (CTA) and neurological disorders resulting from enzyme deficiencies is the inability of active bio-scavengers or catalytically active enzymes to transverse the BBB. Researchers with promising concepts have been forced to abandon research into drugs with high therapeutic potential due to their inability to cross the BBB in therapeutic concentrations (Djupesland et al., 2014). The intranasal route of administration is associated with a number of advantages over systemic administration including rapid CNS bioavailability, early onset of effects and a growing record of success with approved formulations. Intranasal delivery has been the most successful non-invasive, alternative route of entry into the brain for substances that cannot traverse the BBB (Lochhead and Thorne, 2012). An additional advantage of intranasal administration of drugs and proteins to the brain includes the avoidance of first-pass hepatic metabolism. Research into whether the intranasal route might deliver potentially therapeutic amounts of larger biologics such as proteins to the CNS was first described two decade ago (Thorne et al., 1995). Thorne and co-workers proposed that intranasal delivery of solutes to the brain occurs through pathways associated with the olfactory and trigeminal nerves. Solutes applied to the nasal epithelium would be transported to the olfactory bulb and brainstem, respectively, before the distribution to other CNS areas (Thorne et al., 2008, Thorne et al., 2004). This distribution throughout the brain is thought to be accomplished through paravascular spaces between vascular endothelial cells and the surrounding astrocyte sheath which allows bulk flow of fluid and solutes. This system has been termed the glymphatic system because of the involvement of astrocytes and the functional similarities to the lymphatic system (Iliff et al., 2012). Recently, Lochhead et al. (Lochhead et al., 2015) proposed that bulk flow within these paravascular compartments is responsible for the entry of therapeutics to olfactory bulbs and brainstem, and the subsequent distribution to the rest of the brain.
A number of reports have demonstrated protein uptake into brain parenchyma after intranasal administration. Small proteins such as insulin (∼5.8 kD) (Frey, 2013), insulin-like growth factor 1 (∼7.6 kD) (Liu et al., 2001), and nerve growth factor (∼26.5 kD) (Zhu et al., 2011) have been successfully delivered to the brain by the intranasal route, but larger proteins do not gain access to brain parenchyma as readily. Studies comparing the movement of fluorescent tracers with molecular masses of 3 kD and 10 kD showed that compounds within this size range can gain access to brain parenchyma after intranasal administration, and that the use of the extracellular matrix degrading enzyme MMP-9 increased uptake of both compounds into the brain (Lochhead et al., 2015). MMP-9 is a zinc-dependent endopeptidase responsible for both physiological and pathophysiological tissue re-modeling and plays a major role in the degradation of the extracellular matrix. Reports for successful intranasal delivery of active enzymes to brain parenchyma are scanty. One study reported that intranasal delivery of an active enzyme to the brain was accomplished when administering α-l-iduronidase, an 85 kD lysosomal enzyme, to mice deficient in the enzyme (Wolf et al., 2012). Enzyme activity was detectable in the brains of the enzyme deficient mice. Whether the enzyme reached all areas of the brain remained unclear.
Our objective was to provide a simple model system for studying intranasal delivery of enzymes to the brain in their active form. We selected the well-characterized bacterial enzyme chloramphenicol acetyltransferase (CAT), which functions as a trimer with a molecular weight of 75 kD. The mammalian brain does not express significant CAT-like activity (McMahon et al., 1984), making this enzyme a good candidate for intranasal administration and bioavailability studies. We also tested the effect of intranasal pre-treatment with MMP-9 to augment epithelial permeability.
An emerging use for catalytic bio-scavenging enzymes is in the detoxification of organophosphate CTAs as a countermeasure to protect the CNS from excess cholinergic activation after the CTA poisoning (Worek et al., 2014). However, intravenous administration of active organophosphate degrading enzymes will not deliver them to brain parenchyma, thus limiting their efficacy in protecting the CNS. We developed the current method of intranasal delivery of active enzyme to the CNS in order to improve our understanding of enzyme bioavailability via this route with the future goal applying this strategy as an adjunct therapy for CTA poisoning.
Section snippets
Reagents
CAT (Cat no: C2900-25KU) and acetyl-CoA were purchased from Sigma Aldrich (St. Louis, MO). MMP-9 was purchased from Sino Biologicals (Beijing, China). [14C]-labelled chloramphenicol (Cat no: ARC 0401) was obtained from American Radiolabeled Chemicals (St. Louis, MO). High performance thin layer chromatography (HP-TLC) plates were obtained from Merck and phosphor imaging plates were purchased from GE Healthcare Life Sciences (Pittsburgh, PA). Other chemicals were obtained from Sigma Aldrich. The
Results
We have analyzed the levels of CAT enzymatic activity, delivered by intranasal administration, in seven regions of the rat brain by HP-TLC coupled with radiometric assays. We observed four major bands on the HP-TLC plates representing non-acetylated chloramphenicol, two bands of mono-acetylated chloramphenicol due to the acetylation of the 1′ and 3′ carbon positions, as well as di-acetylated chloramphenicol. These findings demonstrate that the active enzyme was transported to the brain where it
Discussion
The aim of this study was to develop a model system for studying the intranasal delivery of active enzymes to the brain with good yield. We chose a bacterial enzyme with resistance to heat inactivation in order to avoid endogenous acetylation activity against chloramphenicol. The nasal mucosa is an advantageous administration route for rapid drug absorption into the CNS. Intranasal brain delivery provides a practical, non-invasive delivery route for therapeutic agents, including higher
Conclusion
Using a sensitive radiometric assay we detected radiolabeled product resulting from CAT enzyme activity in the brain at 15 and 30 min post intranasal administration. We detected enzyme activity throughout the brain, with significantly higher levels of activity observed in some brain areas after pre-treatment with MMP-9. This method can be used to examine the delivery of active enzyme to the brain for a variety of purposes including the possibility of using catalytic bioscavengers to detoxify CNS
Conflict of interest statement
The authors declare no conflicts of interest.
Acknowledgments
We acknowledge Impel NeuroPharma, Inc. (Seattle, WA) for making their intranasal delivery device available for our use. The opinions or assertions contained herein are the private views of the authors, and are not to be construed as reflecting the views of the Department of Defense. This work was supported by the NIH grant NS076448. Graphical abstract adapted from (Swanson, 2004) (http://larrywswanson.com/?page_id=164).
References (23)
- et al.
Permeability issues in nasal drug delivery
Drug Discov Today
(2002) - et al.
Tubule formation by human surface respiratory epithelial cells cultured in a three-dimensional collagen lattice
Am J Physiol
(1993) - et al.
Intranasal treatment of central nervous system dysfunction in humans
Pharm Res
(2013) - et al.
A method for increasing the sensitivity of chloramphenicol acetyltransferase assays in extracts of transfected cultured cells
Anal Biochem
(1987) - et al.
The nasal approach to delivering treatment for brain diseases: an anatomic, physiologic, and delivery technology overview
Ther Deliv
(2014) Intranasal insulin to treat and protect against posttraumatic stress disorder
J Nerv Ment Dis
(2013)- et al.
Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells
Mol Cell Biol
(1982) - et al.
A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta
Sci Transl Med
(2012) Nasal drug delivery—possibilities, problems and solutions
J Control Release
(2003)- et al.
Intranasal administration of insulin-like growth factor-I bypasses the blood-brain barrier and protects against focal cerebral ischemic damage
J Neurol Sci
(2001)
Intranasal delivery of biologics to the central nervous system
Adv Drug Deliv Rev
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