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An into-the-nose (intranasal) formulation of edaravone — the active ingredient in the <a href="https://alsnewstoday.com/what-is-als/">amyotrophic lateral sclerosis</a> (ALS) medication <a href="https://alsnewstoday.com/radicava-edaravone-for-als/">Radicava</a> — may enhance the medication&#8217;s ability to reach brain tissue, according to a recent study.
The formulation, which was made by packaging edaravone into tiny carriers called nanoparticles, was found to ease toxicity in cell cultures, and showed good brain distribution with limited accumulation in other organs when delivered via the intranasal route in mice.
Researchers believe this approach might eventually offer patients an alternate mode of delivery for edaravone with fewer side effects, but could also be used to formulate other <a href="https://alsnewstoday.com/als-treatment/">ALS medications</a> that have a hard time getting into the brain.
&#8220;This study is the first of its kind to develop an ALS drug in a nanoparticulate formulation for nose-to-brain delivery raising hope to ALS patients where currently treatment options are limited,&#8221; researchers wrote.
The study, &#8220;<a href="https://www.sciencedirect.com/science/article/pii/S0168365923003711">Intranasal administration of edaravone nanoparticles improves its stability and brain bioavailability</a>,&#8221; was published in the <a href="https://www.sciencedirect.com/journal/journal-of-controlled-release">Journal of Controlled Release</a>.
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<h3>Radicava designed to combat oxidative stress</h3>
Radicava works to slow functional decline in ALS by combating oxidative stress, a type of cellular damage that&#8217;s implicated in the nerve cell death that characterizes the disease.
It is available to patients in a number of countries as an into-the-vein (intravenous) infusion, but an oral formulation called Radicava ORS is also approved in the <a href="https://alsnewstoday.com/news/fda-approves-radicava-ors-oral-suspension-edaravone-als/">U.S.</a> and <a href="https://alsnewstoday.com/news/radicava-oral-suspension-approved-canada-treat-als/">Canada</a>. Neither formulation has been cleared in the European Union.
Intranasal formulations of medications are thought to offer certain advantages for treating neurological conditions, but such a formulation of Radicava does not currently exist.
With this approach, medications are often packaged into nanoparticles, which have characteristics that help to bypass the selective blood-brain barrier (BBB) and efficiently reach the brain tissue when inhaled through the nose. The BBB is a tight-knit collection of cells that works to keep unwanted molecules out of the brain. A consequence of this is that certain medications, when delivered into the bloodstream, have a hard time getting in.
In addition to helping medications reach the brain in higher concentrations, intranasal therapies are thought to help avoid side effects that arise when a medication accumulates in various organs after a systemic mode of delivery.
[quote=&#8221;center&#8221;]This study is the first of its kind to develop an ALS drug in a nanoparticulate formulation for nose-to-brain delivery raising hope to ALS patients where currently treatment options are limited.[/quote]
<h3>Researchers develop new nanoparticle-encapsulated version of edaravone</h3>
As such, researchers in the U.K. and China developed a new nanoparticle-encapsulated version of edaravone, referred to as NP-EDV and examined its properties in cell cultures and mice.
Results indicated that edaravone could be efficiently loaded into the nanoparticles and had better shelf-life stability than the molecule normally has on its own.
Moreover, NP-EDV exhibited favorable pharmacological properties and reduced signs of oxidative stress and associated cellular toxicity in an oxidative stress cell culture model.
The encapsulated treatment was then tagged with a fluorescent marker and administered to healthy mice via intranasal or intravenous delivery. The fluorescent marker can be imaged to look at the distribution of the medication throughout the body.
While both modes of delivery enabled NP-EDV to reach the brain, similar concentrations were reached with a much lower dose of the intranasal treatment, suggesting &#8220;smaller drug doses can be given without compromising drug concentrations in the brain,&#8221; the researchers wrote.
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<h3>Intranasal route limited uptake of medication largely to brain, GI tract</h3>
On the other hand, distribution of NP-EDV was more widespread throughout the body with the intravenous infusion, showing accumulation in several organs including the liver, whereas the intranasal route limited uptake of the medication largely to the brain and gastrointestinal tract.
&#8220;Altogether the imaging study &#8230; suggested that the [intranasal] route offers a more ‘brain targeting’ approach with relative lower accumulation in the rest of the body,&#8221; the researchers wrote.
Avoiding accumulation in these other organs might help to prevent side effects.
In addition to supporting the potential benefits of an intranasal edaravone formulation, the scientists believe the study&#8217;s findings have broader applications. The nanoparticles they developed could also be used to package other potential ALS therapies that have a limited ability to reach the brain on their own.
&#8220;The current approach also opens up new opportunities for repurposed drugs for treatment of ALS which could otherwise be limited due to poor BBB crossing and brain bioavailability,&#8221; the team concluded.

Intranasal formulation helps edaravone reach brain: Early study

An immune protein that is mostly produced in neurons was found to drive damage to mitochondria, which produce energy for cells, and nerve fiber loss, a study has found.
Suppressing the protein, called gasdermin-E (GSDME), prevented nerve fiber loss in cells derived from people with <a href="https://alsnewstoday.com/what-is-als/">amyotrophic lateral sclerosis</a> (ALS), and also prolonged survival and improved motor function in an ALS mouse model.
Researchers noted that understanding the details of GSDME-mediated nerve damage may support the development of new <a href="https://alsnewstoday.com/als-treatment/">therapeutic strategies</a> for neurodegenerative diseases.
“The unmet need for therapies for neurodegenerative diseases is huge, and our work opens up a whole new pathology that we could address,” Judy Lieberman, MD, PhD, professor at Harvard Medical School (HMS) and study co-lead said in a <a href="https://hms.harvard.edu/news/new-insights-als">university press release</a>.
“We describe a pathway and molecules that you can target for treating many neurodegenerative diseases,” said Lieberman, who is also chair of cellular and molecular medicine at Boston Children’s Hospital.
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The discovery was described in the study “<a href="https://www.cell.com/neuron/fulltext/S0896-6273(23)00123-X?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS089662732300123X%3Fshowall%3Dtrue">Gasdermin-E mediates mitochondrial damage in axons and neurodegeneration</a>,” which was published in the journal <a href="https://www.cell.com/neuron/home">Neuron</a>.
ALS is marked by the shrinkage and death of motor neurons, the nerve cells that send messages between the nervous system and muscles to control voluntary movements. In neurological diseases such as ALS, nerve cell death is often preceded by the impaired function of mitochondria and the loss of nerve fibers called axons.
<h3>Gasdermins are proteins linked to cell death</h3>
Gasdermins (GSDMs) are a family of proteins linked to cell death via a process called pyroptosis — a form of death triggered by the formation of pores in the cell membrane. Pyroptosis occurs <a href="https://jeccr.biomedcentral.com/articles/10.1186/s13046-021-01959-x">most often upon infection</a> and is thought to be part of the antimicrobial response, but it has also been associated with disease-related inflammation.
“Inflammation is a double-edged sword and could be either protective or destructive based on context,” said Isaac Chiu, PhD, associate professor of immunology at HMS and study co-lead.
GSDME is a GSDM family member produced in the brain and spinal cord, but its role in nerve cell function remains largely unknown. The team at Harvard set out to address that question.
The protein was found to be activated when neurons were exposed to chemicals that are toxic to mitochondria to model features of neurodegeneration. GSDME specifically targeted the membranes within mitochondria, where it caused pores that released the mitochondrial content to mediate neuronal damage.
Consistently, mouse neurons modified to lack GSDME showed greater mitochondrial integrity and resistance to the toxic chemicals. Overall, evidence indicated that GSDME regulated the integrity of axons, and when activated locally, it destroyed mitochondria and damaged axons without affecting neuron cell bodies.
“If you look at a plate of neurons, you see a jungle of axons,” said Himanish Basu, PhD, co-first author of the study. “But if you look at a plate where gasdermin E is activated, you see retractions of these cellular processes.”
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<h3>GSDME contributed to mitochondrial dysfunction, axon loss</h3>
To understand whether GSDME could contribute to the neuronal death caused by ALS-associated proteins, the team grew mouse and human-derived neurons with TDP43 and PR-50 — a product of C9ORF72 mutations. This exposure induced GSDME activation and caused mitochondrial dysfunction and axon loss.
In contrast, exposure to these proteins did not impact axons in cells lacking GSDME. The team noted these results mirrored those obtained using mitochondrial toxins.
“Our work is an example of how immunology can help explain neurodegeneration on a mechanistic level, and what drives axon loss and neuronal injury,” said Dylan Neel, a graduate student in Chiu’s lab and a co-first author.
Finally, the team tested the impact of GSDME in a standard ALS mouse model, which developed progressive loss of motor neurons in the spinal cord. The researchers detected activated GSDME in the spinal cords of symptomatic ALS mice, increasing further by the late stages of the disease, but the protein was not found in pre-symptomatic animals.
Deleting GSDME in ALS mice improved motor function, prevented axon and motor neuron loss, and reduced nerve-related inflammation, which ultimately extended the animals&#8217; survival.
“We identify GSDME as an executioner of neuronal mitochondrial dysfunction that may contribute to neurodegeneration,” the researchers wrote. “Future work should determine the feasibility of targeting GSDME expression or function.”
Chiu added: “We revealed an innate immune molecule playing a role in neurodegeneration, which presents a new avenue of thinking about neuronal health.”

This immune protein plays key role in nerve damage, early study finds

A CRISPR/Cas9 gene editing system can be used to remove the repeat expansion in the C9ORF72 gene, the most common genetic cause of <a href="https://alsnewstoday.com/what-is-als/">amyotrophic lateral sclerosis</a> (ALS), a new study reports.
The system showed positive proof-of-concept effects in mouse models and in human cells, reducing the amount of faulty RNA molecules and small proteins that contribute to nerve cell death in ALS. RNA is the intermediate molecule produced from a DNA sequence that serves as a template for protein production.
The study, &#8220;<a href="https://www.nature.com/articles/s41467-022-33332-7">CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro</a>,&#8221; was published in <a href="https://www.nature.com/ncomms">Nature Communications</a>.
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<h3>ALS-causing C9ORF72 mutations involve abnormal repeats of DNA sequence</h3>
The <a href="https://alsnewstoday.com/causes-of-als/">causes of ALS</a> are not completely understood, but some cases are associated with <a href="https://alsnewstoday.com/als-and-genetics/">genetic mutations</a>. Mutations in a gene called C9ORF72 are the most common genetic cause of ALS, found in about 40% of <a href="https://alsnewstoday.com/forms-of-als/familial-als/">familial</a> cases and 5% of <a href="https://alsnewstoday.com/forms-of-als/sporadic-als/">sporadic</a> cases.
ALS-causing mutations in C9ORF72 mainly involve an abnormal repeat of a certain sequence of DNA, called an hexanucleotide repeat expansion (HRE). In the gene, there is a region where six nucleotides (the &#8220;letter&#8221; of the genetic code), namely GGGGCC, are repeated multiple times. In healthy versions of the C9ORF72 gene, there are about two dozen repeats, but in ALS-causing mutations, there can be hundreds or even thousands of these repeats.
The HRE interferes with production of the C9ORF72 protein and leads to the formation of toxic RNA intermediate molecules and small proteins, all of which is thought to contribute to the progression of ALS.
CRISPR/Cas9 was developed by manipulating a similar system that bacteria use to defend themselves against viral infection. Very simplistically, CRISPR/Cas9 gene editing can be used to &#8220;cut out&#8221; a specific region of DNA.
[quote=&#8221;center&#8221;]These proof-of-concept findings support the view that CRISPR methodologies should be explored as a strategy to halt or slow disease progression in [ALS] patients, and potentially other disorders caused by expanded domains of repeated DNA[/quote]
In the study, researchers developed a CRISPR/Cas9 system to &#8220;cut out&#8221; the HRE in an ALS-mutant version of the C9ORF72 gene, without affecting the parts of the gene that provide instructions for making the C9ORF72 protein.
To deliver the system to cells, it was packaged in adeno-associated virus (AAV) vectors. AAV is commonly used in gene therapy applications because it can effectively deliver genetic cargo to human cells and is easy to manipulate in a laboratory, but it doesn&#8217;t cause notable illness in people.
&#8220;AAV has many advantages and is presently regarded as the safest and most effective gene therapy vector for the [brain and spinal cord], with potentially a long-lived therapeutic effect following a single treatment,&#8221; the researchers wrote. However, they stressed that the viral vector they used was &#8220;merely a delivery tool for a proof-of-mechanism,&#8221; noting that other delivery tools will likely be better suited for clinical applications.
After designing the system and conducting initial proof-of-concept and refinement tests in cell lines, the researchers tested the system in several models of ALS.
First, in a battery of experiments done in mouse cell models, the researchers demonstrated that the system could efficiently remove the HRE from nerve cells harboring the ALS-causing mutation in C9ORF72. As designed, the CRISPR/Cas9 system did not interfere with production of the C9ORF72 protein, but it markedly reduced the production of toxic intermediate molecules made from the HRE.
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<h3>CRISPR/Cas9 was able to reduce production of toxic molecules</h3>
Additional experiments done in three mouse models of ALS yielded generally consistent results, with the system effectively removing the HRE and decreasing toxic intermediate production, without affecting C9ORF72 protein levels.
Of note, because the CRISPR/Cas9 system was relatively large, it needed to be delivered to the mice using two separate AAV vectors. Analyses showed that most of the mice&#8217;s cells were delivered genetic cargo from both vectors.
In a final set of experiments, the researchers tested the system in human nerve cells derived from people with ALS caused by C9ORF72 mutations. Consistent with the mouse experiments, results showed that the system could reduce the production of toxic molecules without interfering with C9ORF72 protein levels — in fact, data suggested the system may increase C9ORF72 protein expression in human cells.
&#8220;These proof-of-concept findings support the view that CRISPR methodologies should be explored as a strategy to halt or slow disease progression in [ALS] patients, and potentially other disorders caused by expanded domains of repeated DNA,&#8221; the researchers concluded.

Tag: preclinical study

Intranasal formulation helps edaravone reach brain: Early study

This immune protein plays key role in nerve damage, early study finds

CRISPR/Cas9 Gene Editing Used to ‘Cut Out’ ALS Mutation: Early Study