Nanoparticle research tested in locusts focuses on new drug-delivery method

Delivering life-saving drugs directly to the brain in a safe and effective way is a challenge for medical providers. One key reason: the blood-brain barrier, which protects the brain from tissue-specific drug delivery. Methods such as an injection or a pill aren’t as precise or immediate as doctors might prefer, and ensuring delivery right to the brain often requires invasive, risky techniques.

A team of engineers from Washington University in St. Louis has developed a new nanoparticle generation-delivery method that could someday vastly improve drug delivery to the brain, making it as simple as a sniff.

“This would be a nanoparticle nasal spray, and the delivery system could allow a therapeutic dose of medicine to reach the brain within 30 minutes to one hour,” said Ramesh Raliya, research scientist at the School of Engineering & Applied Science.

“The blood-brain barrier protects the brain from foreign substances in the blood that may injure the brain,” Raliya said. “But when we need to deliver something there, getting through that barrier is difficult and invasive. Our non-invasive technique can deliver drugs via nanoparticles, so there’s less risk and better response times.”

The novel approach is based on aerosol science and engineering principles that allow the generation of monodisperse nanoparticles, which can deposit on upper regions of the nasal cavity via diffusion. Working with Assistant Vice Chancellor Pratim Biswas, chair of the Department of Energy, Environmental & Chemical Engineering and the Lucy & Stanley Lopata Professor, Raliya developed an aerosol consisting of gold nanoparticles of controlled size, shape and surface charge. The nanoparticles were tagged with fluorescent markers, allowing the researchers to track their movement.

Next, Raliya and biomedical engineering postdoctoral fellow Debajit Saha exposed locusts’ antennae to the aerosol, and observed the nanoparticles travel from the antennas up through the olfactory nerves. Due to their tiny size, the nanoparticles passed through the brain-blood barrier, reaching the brain and suffusing it in a matter of minutes.

The team tested the concept in locusts because the blood-brain barriers in the insects and humans have anatomical similarities, and the researchers consider going through the nasal regions to neural pathways as the optimal way to access the brain.

“The shortest and possibly the easiest path to the brain is through your nose,” said Barani Raman, associate professor of biomedical engineering. “Your nose, the olfactory bulb and then olfactory cortex: two relays and you’ve reached the cortex. The same is true for invertebrate olfactory circuitry, although the latter is a relatively simpler system, with supraesophageal ganglion instead of an olfactory bulb and cortex.”

To determine whether or not the foreign nanoparticles disrupted normal brain function, Saha examined the physiological response of olfactory neurons in the locusts before and after the nanoparticle delivery. Several hours after the nanoparticle uptake, no noticeable change in the electrophysiological responses was detected.

“This is only a beginning of a cool set of studies that can be performed to make nanoparticle-based drug delivery approaches more principled,” Raman said.

The next phase of research involves fusing the gold nanoparticles with various medicines, and using ultrasound to target a more precise dose to specific areas of the brain, which would be especially beneficial in brain-tumor cases.

“We want to drug target delivery within the brain using this non-invasive approach,” Raliya said. “In the case of a brain tumor, we hope to use focused ultrasound so we can guide the particles to collect at that particular point.”

 

Less fear: How LSD affects the brain

Scientists at the University of Basel have shown that LSD reduces activity in the region of the brain related to the handling of negative emotions like fear. The results, published in the scientific journal Translational Psychiatry, could affect the treatment of mental illnesses such as depression or anxiety.

Hallucinogens have many different effects on the psyche; among other things, they alter perception, thought, and temporal and emotional experience. After the Basel-based chemist Albert Hofmann discovered lysergic acid diethylamide (LSD) in the 1940s, there was a huge amount of interest in the substance, particularly in psychiatry. It was hoped, for example, that it could provide insights into the development of hallucinations, and studies were conducted on its effectiveness on illnesses such as depression or alcohol dependency. In the 1960s, LSD was declared illegal worldwide, and medical research on it came to a standstill.

In the last few years, however, interest in researching hallucinogens for medical purposes has been revived. Psychoactive substances such as LSD, particularly in combination with psychotherapies, could offer an alternative to conventional medication. It is now known that hallucinogens bind to a receptor of the neurotransmitter serotonin; how the changes of consciousness influence the activity and connectivity of the brain, however, is not yet known.

LSD alters brain activity

Researchers at the University Psychiatric Clinics (UPK) and the Department of Pharmacology and Toxicology at the University Hospital Basel (USB) have now conducted a study into the acute effect of LSD on the brain. They used functional magnetic resonance imaging (fMRI) to measure the brain activity of 20 healthy people after taking 100 micrograms of LSD. During the MRI scan, the participants were shown images of faces portraying different emotional states such as anger, joy or fear.

Professor Stefan Borgwardt and his team showed that the depiction of fear under LSD led to a notably lower level of activity in the amygdala — an area of the brain that is believed to be central to the processing of emotions. This observation could explain some of the changes in emotional experience that occur after taking hallucinogens.

Less fear after taking LSD

In a second step, the researchers, together with clinical pharmacologists at the University Hospital Basel, examined whether the subjective experience altered by LSD is associated with the amygdala. This appears to be the case: the lower the LSD-induced amygdala activity of a subject, the higher the subjective effect of the drug. “This ‘de-frightening’ effect could be an important factor for positive therapeutic effects,” explains Doctor Felix Müller, lead author of the study. The researchers presume that hallucinogens may cause many more changes in brain activity. Further studies will investigate this, with a particular focus on their therapeutic potential.

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Making a ‘beeline’ past the blood-brain barrier for drug delivery

Most medicines can’t get through the blood-brain barrier (BBB), a highly selective membrane that separates the circulatory system from the fluid bathing the brain. Certain peptides in animal venoms, however, can navigate across it to inflict damage. Now, researchers are capitalizing on venomous sneak attacks by developing a strategy based on a bee-venom peptide, apamin, to deliver medications to the brain.

The researchers will present their work today at the 253rd National Meeting & Exposition of the American Chemical Society (ACS). ACS, the world’s largest scientific society, is holding the meeting here through Thursday. It features more than 14,000 presentations on a wide range of science topics.

“We thought that because the venoms of some animals are able to attack the central nervous system, they should be able to go through the blood-brain barrier and possibly shuttle drugs across it,” Ernest Giralt, Ph.D., says. Apamin is known to accumulate in the central nervous system of people who’ve been stung by bees.

But the idea of using the apamin peptide itself had some drawbacks. “We knew we could not use apamin directly because it’s toxic,” he says. “But the good news is that the origin of the toxicity is well-known. We thought we could probably modify apamin in such a way that the toxicity would be eliminated, but it would still keep its ability to act as a transporter.”

Apamin’s toxicity stems from its interactions with a potassium channel in neurons. A positively charged group in the apamin molecule mimics the potassium ion and blocks the potassium channel when it binds. To eliminate the toxicity, Giralt’s group at the Institute for Research in Biomedicine (IRB Barcelona, Spain) removed the positively charged chemical anchor that attaches apamin to the channel. Then, the researchers checked to make sure the molecule could still cross the BBB. “This modification made apamin much less toxic, and its ability to cross the BBB was intact,” Giralt says. “This was very good news.”

As a next step, the researchers started tinkering with the molecule to make it smaller and also to make it invisible to the immune system to reduce potential side effects. Several versions of apamin later, they ended up with a promising version called Mini-Ap4. “It surprised us that this molecule crossed the blood-brain barrier much better than apamin itself — it was pure serendipity,” Giralt says. Mini-Ap4 also did not trigger a strong immune system response in animal models, an important factor in drug design.

Other BBB shuttles are in development, but many of them are based on linear peptides, which can be degraded by proteases before a medicine makes it to the brain. “Our niche is that our peptides are cyclic, or in a ring structure, making them completely resistant to proteases,” Giralt explains.

After these initial studies, the team will then put Mini-Ap4 to work, testing two different shuttling strategies. The first will be to simply attach Mini-Ap4 to a protein with a chemical bond and see if it can carry the cargo across the BBB. The second approach will involve filling a nanoparticle with medication and coating the nanoparticle with a forest of Mini-Ap4 molecules to facilitate the transfer across the BBB. The researchers will investigate these strategies in human cells and in mice.

In other preliminary work, the researchers discovered that their version of apamin actually has two conformations, or shapes, and the team is using nuclear magnetic resonance spectroscopy to figure out which one is biologically active. “With that knowledge, we could design even better analogs,” Giralt says. He adds that a person who is allergic to bees probably wouldn’t be allergic to Mini-Ap4, but more work is needed to fully address this issue.

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Experimental small molecule shows potential in preventing meth relapse

New research from The Scripps Research Institute (TSRI) suggests that the reason methamphetamine users find it so hard to quit — 88 percent of them relapse, even after rehab — is that meth takes advantage of the brain’s natural learning process. The TSRI study in rodent models shows that ceasing meth use prompts new neurons to form in a brain region tied to learning and memory, suggesting that the brain is strengthening memories tied to drug-seeking behavior.

“New neuronal growth is normally thought of as a good thing, but we captured these new neurons assisting with ‘bad’ behaviors,” said Chitra Mandyam, who led the research as an associate professor at TSRI before starting a new position at the Veterans Medical Research Foundation and the University of California, San Diego.

The scientists discovered that they could block relapse by giving animals a synthetic small molecule to stop new neurons from forming. This molecule, called Isoxazole-9 (Isx-9), also appeared to reverse abnormal neuronal growth that developed during meth use.

The new research was published this week in the journal Molecular Psychiatry.

Young Neurons Gone Bad

Neurons are born all the time in a process called neurogenesis. In a 2010 study, Mandyam and her colleagues at TSRI showed that increased neurogenesis is tied to a higher risk of drug relapse, but they weren’t sure of the new neurons’ role in the process. The researchers were especially curious about a “burst” of neurogenesis that occurs during abstinence from meth.

The new study may explain why the brain is so eager to make neurons during abstinence: meth hijacks the natural neurogenesis process.

Normally, new neurons help us learn by forming new circuits to connect rewards, like food, to reward-associated memories. For example, we learn early on that the refrigerator holds food. “In a non-drug environment, this is a healthy process,” said Mandyam. But the brain isn’t good at separating healthy rewards from the dangerous high of drug use.

Using rat models of meth addiction, the researchers showed that forced abstinence prompted the development of new neurons called granule cell neurons in a brain region called the dentate gyrus, which is associated with memory formation. These new neurons drove compulsive-like drug seeking and relapse by strengthening drug-associated memories. The rats learned to associate a particular location in their environment with meth use. Returning to this location during abstinence later served as a triggering cue — prompting a recovering addict to relapse.

A Potential Way to Stop Relapse

Next, the researchers tested whether the synthetic small molecule Isx-9 could inhibit this process. Previous studies had shown that Isx-9 could block cell division of some types of cells, but it had not been tested as a way to block neurogenesis and fight meth relapse. Working closely with Professor Kim Janda’s lab at TSRI, which supplied the molecule, Mandyam and her colleagues found that meth-addicted rats given Isx-9 during abstinence were less likely to relapse into drug use. Isx-9 indeed blocked neurogenesis, appearing to keep their brains from strengthening drug-associated memories. For these rats, the environment where they took the drug was no longer a strong trigger for relapse.

Interestingly, the researchers only saw the benefits of Isx-9 in rats that were “high responders” to meth. From the beginning of the experiment, some of the rats were simply not as interested in the drug — Mandyam called them the “casual users.” “Just like humans, animals also show remarkable individual differences in drug seeking,” said Mandyam. She plans to further study these individual differences to better understand how to address addiction and recovery.

Isx-9 also appears to repair some of the structural changes seen in neurons exposed to meth. In high-responder rats, Isx-9 restored the neuronal structures crucial for normal cell signaling.

The researchers also plan to further investigate potential side effects of Isx-9, and Mandyam hopes future studies will set the stage to test Isx-9 in clinical trials for meth addiction.

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Hair testing shows high prevalence of new psychoactive substance use

In the last decade hundreds of new psychoactive substances (NPS) have emerged in the drug market, taking advantage of the delay occurring between their introduction into the market and their legal ban. According to the Drug Enforcement Agency (DEA) NPS describes a recently emerged drug that may pose a public health threat. The DEA issues a quarterly Emerging Threat Report, which catalogues the newest identified NPS.

NPS tend to mimic the psychotropic effects of traditional drugs of abuse, but their acute and chronic toxicity, and side-effects are largely unknown. While seizure data from the DEA is often used to indicate what new drugs are being sold in the US, there is a lack of research examining and confirming who has been using such drugs.

Joseph J. Palamar, PhD, MPH, a New York University researcher, has been researching incidental and intentional use of NPS by young adults. His current line of inquiry has focused on survey methods, qualitative interviews, and hair sampling to ascertain frequency and type of NPS use by nightclub-goers — a demographic which traditionally has a relaxed view towards recreational drug experimentation and use.

NPS are common adulterants in drugs such as ecstasy (MDMA), which has seen an increase in popularity since it became marketed as “Molly.” Ironically, “Molly” connotes a product that is pure MDMA. In a related study, Palamar and his team found that four out of ten nightclub/festival attendees who used ecstasy or “Molly” tested positive for “bath salts” despite reporting no use.

In their current study, “Hair Testing for Drugs of Abuse and New Psychoactive Substances in a High-Risk Population,” Dr. Alberto Salomone, an affiliated researcher at the Centro Regionale Antidoping e di Tossicologia “A. Bertinaria,” Orbassano, Turin, Italy and Dr. Palamar, affiliated with NYU’s Center for Drug Use and HIV Research (CDUHR), collected hair samples from 80 young adults outside of New York City nightclubs and dance festivals, from July through September of 2015. Hair samples from high-risk nightclub and dance music attendees were tested for 82 drugs and metabolites (including NPS) using ultra-high performance liquid chromatography-tandem mass spectrometry.

“Hair analysis represents a reliable and well-established means of clinical and forensic investigations to evaluate drug exposure, said Dr. Salomone. “Hair is the most helpful specimen when either long-time retrospective information on drug consumption is of interest.” “Most NPS can no longer be detected in urine, blood, or saliva within hours or days after consumption, but hair is particularly beneficial because many drugs can be detected months after use.”

Of the eighty samples, twenty-six tested positive for at least one NPS — the most common being a “bath salt” (synthetic cathinone) called butylone (present in twenty-five samples). The “bath salts” methylone and even alpha-PVP (a.k.a.: “Flakka”) were also detected. The researchers find the presence of Flakka alarming as this drug has been associated with many episodes of erratic behavior and even death in Florida. Other new drugs detected included new stimulants called 4-FA and 5/6-APB.

“We found that many people in the nightclub and festival scene have been using new drugs and our previous research has found that many of these people have been using unknowingly,” said Dr. Palamar, also an assistant professor of Population Health at NYU Langone Medical Center (NYULMC).

Hair analysis proved a powerful tool to Drs. Salomone and Palamar and their team, allowing them to gain objective biological drug-prevalence information, free from possible biases of unintentional or unknown intake and untruthful reporting of use.

“Such testing can be used actively or retrospectively to validate survey responses and inform research on consumption patterns,” notes Dr. Palamar.

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Potential drugs and targets for brain repair

Researchers have discovered drugs that activate signaling pathways leading to specific adult brain cell types from stem cells in the mouse brain, according to a study publishing on 28 March in the open access journal PLOS Biology by Kasum Azim of the University of Zurich and colleagues from INSERM/university of Lyon and University of Portsmouth. The results may open new avenues for drug development aimed at treatment of degenerative brain disorders.

New neurons, and support cells called oligodendrocytes, arise during development throughout adulthood from neural stem cells in the subventricular zone, a region of the forebrain adjacent to the ventricles. The transcriptional changes associated with the development of each cell type in the newborn mouse have been catalogued in publicly accessible databases. Similarly, the transcriptional changes produced by thousands of chemicals approved for clinical use have also been catalogued. In the new study, the authors used these databases (which included their own previously generated data) to find overlaps between transcriptional changes associated with cell differentiation and drug treatments, on the premise that these might identify potential therapies to reverse neurodegenerative diseases.

Toward that end, they characterized differences in signaling pathways in “microdomains” of the subventricular zone where neurons or oligodendrocytes get their start in life. They found several potentially important differences between neuron-specific and oligodendrocyte-specific microdomains, and used these findings to identify similar changes in gene expression in the small molecule drug database.

That led them to a set of small molecule drugs whose transcriptional signatures were similar to those of either neuronal or oligodendrocytic development. They showed that one such molecule, called LY-294002 specifically enhanced normal oligodendrogenesis from neural stem cells in newborn mice. In adult mice, different molecules (AR-A014418 and CHIR99021) counteracted the gradual loss of neurogenic capacity and lineage diversity of the adult subventricular zone. Finally, this later molecule promoted robust regeneration of oligodendrocytes and a smaller increase in neurons in a model of perinatal hypoxic brain injury.

These results may be valuable in several ways. First, because the small molecule drug data point to important cellular pathways, they provide new insights into the mechanisms of neural development and repair, which can be exploited to develop new strategies for treatment. Second, they identify several new drugs, each already approved for clinical use, whose therapeutic potential for brain injury repair can now be explored. Finally, they provide a proof-of-principle for a novel approach to identify other potentially valuable new drugs that can directly affect neural regeneration, and that may be developed for treating brain diseases.

“Controlling the fate of neural stem cells is a key therapeutic strategy in regenerative medicine,” said Azim and coworkers. “The strategy outlined in this study may allow us to quickly identify multiple drug candidates and get them into the drug development pipeline, where their potential as treatments can then be further assessed.”

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Caffeine boosts enzyme that could protect against dementia

A study by Indiana University researchers has identified 24 compounds — including caffeine — with the potential to boost an enzyme in the brain shown to protect against dementia.

The protective effect of the enzyme, called NMNAT2, was discovered last year through research conducted at IU Bloomington. The new study appears today in the journal Scientific Reports.

“This work could help advance efforts to develop drugs that increase levels of this enzyme in the brain, creating a chemical ‘blockade’ against the debilitating effects of neurodegenerative disorders,” said Hui-Chen Lu, who led the study. Lu is a Gill Professor in the Linda and Jack Gill Center for Biomolecular Science and the Department of Psychological and Brain Sciences, a part of the IU Bloomington College of Arts and Sciences.

Previously, Lu and colleagues found that NMNAT2 plays two roles in the brain: a protective function to guard neurons from stress and a “chaperone function” to combat misfolded proteins called tau, which accumulate in the brain as “plaques” due to aging. The study was the first to reveal the “chaperone function” in the enzyme.

Misfolded proteins have been linked to neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases, as well as amyotrophic lateral sclerosis, also known as ALS or Lou Gehrig’s disease. Alzheimer’s disease, the most common form of these disorders, affects over 5.4 million Americans, with numbers expected to rise as the population ages.

To identify substances with the potential to affect the production of the NMNAT2 enzyme in the brain, Lu’s team screened over 1,280 compounds, including existing drugs, using a method developed in her lab. A total of 24 compounds were identified as having potential to increase the production of NMNAT2 in the brain.

One of the substances shown to increase production of the enzyme was caffeine, which also has been shown to improve memory function in mice genetically modified to produce high levels of misfolded tau proteins.

Lu’s earlier research found that mice altered to produce misfolded tau also produced lower levels of NMNAT2.

To confirm the effect of caffeine, IU researchers administered caffeine to mice modified to produce lower levels of NMNAT2. As a result, the mice began to produce the same levels of the enzyme as normal mice.

Another compound found to strongly boost NMNAT2 production in the brain was rolipram, an “orphaned drug” whose development as an antidepressant was discontinued in the mid-1990s. The compound remains of interest to brain researchers due to several other studies also showing evidence it could reduce the impact of tangled proteins in the brain.

Other compounds shown by the study to increase the production of NMNAT2 in the brain — although not as strongly as caffeine or rolipram — were ziprasidone, cantharidin, wortmannin and retinoic acid. The effect of retinoic acid could be significant since the compound derives from vitamin A, Lu said.

An additional 13 compounds were identified as having potential to lower the production of NMNAT2. Lu said these compounds are also important because understanding their role in the body could lead to new insights into how they may contribute to dementia.

“Increasing our knowledge about the pathways in the brain that appear to naturally cause the decline of this necessary protein is equally as important as identifying compounds that could play a role in future treatment of these debilitating mental disorders,” she said.

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