Neurotrophins and their receptors are very old molecules
Neurotrophins (NT’s) are proteins made in the nervous system of mammals, including humans, and in vertebrates as old in the animal family tree as fishes and birds. NT’s are essential for normal brain development, and in the adult brain NT’s protect nerve cells (neurons) from dying due to a variety of stresses. The protein receptors for NT’s, known as “tyrosine kinases” (Trk, pronounced “trek”) are also very old molecules.
Although NT’s and their Trk receptors have been in the brains of creatures on the earth for millions of years, we’ve known about them for only a few decades. Nerve growth factor, NGF, was discovered in 1956 by the Italian neurologist Rita Levi-Montalcini, who won a Nobel Prize for her work. Since then, scientists have identified several other NT’s, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), NT3, and NT5/6.
All of these NT’s appear in the developing brain at various times, and some like NGF and BDNF persist into adulthood. The Trk receptors for NT proteins also can be found in the adult brain, providing the potential for stimulating beneficial, life-prolonging NT effects on neurons if the Trk receptors could only be activated.
DHEA is also an old molecule that can activate Trk receptors
Dehydroepiadrosterone (DHEA) is another biologically very old chemical, in this case a small molecule known as a “steroid precursor” that is made in the brains of many animals. DHEA serves multiple roles, including being a precursor for sex steroids (estrogen, testosterone), but it also has the interesting capacity to bind to and activate Trk receptors for NT proteins. However, its capacity for binding to and activating Trk receptors is reduced compared to its other known functions.
Microneurotrophins (MNT’s) are DHEA analogues with potent Trk receptor properties
Because of their large size, NT proteins enter the brain poorly and are not useful as therapies for brain diseases. Normally they are manufactured inside the brain, and crossing the blood-brain barrier (BBB) is not an issue. Over the last decade, Dr. Achilleas Gravanis and his colleagues at the University of Crete have synthesized and studied multiple DHEA analogues, known as MNT’s. These small molecules activate potently Trk receptors for NT proteins, particularly TrkA (the receptor for NGF) and TrkB (the receptor for BDNF). MNT’s have the potential for use as small molecule drug therapies to stimulate Trk receptors in the brain and provide all the desirable health- and survival promoting benefits of large NT proteins that cannot readily cross the BBB. MNT’s also do not have any sex steroid properties.
MNT’s show promise for treating diseases of the aging brain
The neurons we are born with must last a lifetime. That happens because with rare exception, neurons do not divide as do other cells in the body, like cells in our skin, bone marrow and gastrointestinal system. Neurons are thus “post-mitotic”, meaning removed from cell division (mitosis). They are not easily replaced, and they also require large amounts of energy to function.
As we age, our neurons show the ravages of time, mainly resulting from the inefficiency of energy production with increased damage due to oxygen free radicals. This impairment of energy production leads to more free radicals, which then stimulates brain inflammation, resulting in a death spiral for neurons that NT’s (and MNT’s) can block at multiple levels.
MNT’s have been shown to inhibit all the major known events leading to neuron demise, including biochemical cell death pathways. As a result, MNT’s have great promise for reversing the major brain diseases of aging, such as Alzheimer’s (AD) and Parkinson’s (PD) that afflict millions of Americans.
MNT’s show great promise for ALS
Although many scientists have worked devotedly for decades, we still do not have any cell or animal models that reliably predict drug effectiveness for humans with diseases such as AD, PD or ALS. While frustrating, this problem requires creative approaches to predict drug benefits.
Since MNT’s have many survival promoting actions for neurons, and since in ALS the death of motor neurons is responsible for the major symptoms leading to disability and death, we have focused on enhancing survival of motor neurons with three approaches:
- Our three MNT analogues all showed effectiveness in reducing neuronal stress in a zebrafish model of genetic ALS. Zebrafish are vertebrates with a short lifespan that allows rapid screening of drug effects. The zebrafish used have a fluorescent protein made in neurons that experience activation of a cell stress (heat-shock protein) response. BNN27 was the most effective MNT tested and reduced the stress response >50%. None of the MNT’s caused sedation. (Drs Chris Binney, Tennore Ramesh and Pamela Shaw, Co-PI’s, University of Sheffield, Sheffield Institute for Translational Neuroscience, Sheffield, U.K.)
- BNN27 was consistently effective in increasing survival of mouse motor neurons co-cultured with glial cells (astrocytes) derived from humans with genetic (C9orf72, mSOD1) or sporadic ALS (sALS-1, sALS-2). ALS astrocytes (the major cell in the brain) produce neurotoxic chemicals that kill normal motor neurons, and ALS may arise as a result of these astrocyte neurotoxins. In some test systems BNN27 was the most effective MNT tested, and overall BNN27 was the most neuroprotective MNT examined. (Drs. Laura Ferraiuolo and Pamela Shaw, Co-PI’s, University of Sheffield, Sheffield Institute for Translational Neuroscience, Sheffield, U.K.
- In human motor neurons derived from both embryonic and induced stem cells, BNN27 and BNN23 most resembled NGF and BDNF using next-generation sequencing of genes. These results that examined expression of over 9300 human motor neuron genes support use of BNN27 and BNN23 in ALS. (Drs. Laura O’Brien, David Brohawn and James Bennett, Co-PI’s, Virginia Commonwealth University and Neurodegeneration Therapeutics, Inc., Richmond/Charlottesville, VA)
MNT’s have high permeability to the BBB and will not be pumped out of the brain
For a drug to be effective in treating brain diseases, it must both enter the brain and remain in the brain by not being pumped back out by specific transporters. That means the drug must cross the BBB and not be removed from the brain and pumped back into the blood. All three MNT candidates have been tested for permeability using both artificial and natural cell membranes, and as substrates for being pumped out of the brain by specific transporters. MNT drugs were found to have high BBB permeability and to not be substrates for pumping out of the brain. These results are very encouraging for use of MNT’s to treat brain diseases.
The lead candidate MNT BNN27 is slowly metabolized by human liver but very rapidly metabolized by mouse liver
Most drugs are chemically modified (metabolized) by the liver. The drug metabolites produced can be “active”, meaning they mimic desirable properties of the parent drug, “inactive”, or in rare cases “toxic”, meaning they can cause organ damage. The FDA requires that the primary metabolites of any drug be identified so that their activity and toxicity can be determined.
So far the lead MNT BNN27 has been incubated with liver cells from mice or humans. With mouse liver cells BNN27 was rapidly changed within minutes into at least 7 primary metabolites, whereas with human liver cells, BNN27 was much more slowly changed over hours into 2 or possibly 3 metabolites. We are in the process of identifying the chemical structures of major metabolites of BNN27 exposed to human liver cells, having them synthesized, and determining their activities and/or toxicities. These findings also indicate that any further testing of BNN27 in mice will be complicated by their rapid liver metabolism into multiple metabolite species. For this reason we are focusing our efforts on MNT actions in non-rodent models.
The MNT BNN27 is now ready for safety pharmacology (toxicology) testing prior to seeking an IND
Before any drug can be given to humans, the FDA requires extensive toxicity testing in animals. This usually takes the form of administering the drug to at least two divergent species (ie, rodent and dog), followed by close clinical observation, blood testing and autopsy examination of all internal organs. If the drug is non-toxic at doses predicted to be similar to those given to humans, then this information in combination with “pre-clinical” data (outlined above in 1-7) is presented to the FDA as the process of obtaining an “Investigation New Drug” (IND) permit. The IND allows a new drug to be experimentally administered to humans under the auspices of protocol(s) approved by the FDA. Drugs administered under IND’s are regulated by the FDA and may not be sold
As might be predicted, the major initial concern of the FDA is for toxicity to humans. Early clinical studies cannot focus on drug efficacy, rather they must focus on potential organ toxicity. As a result, the initial study populations are small, and an experimental drug even for such a devastating condition as ALS must be initially tested in limited populations. If toxicity does not appear, or is manageable, then the FDA will allow studies of larger populations. Typically 1-2 years of early testing are required before larger populations are studied, and “compassionate” use of drug is allowed.
Safety pharmacology testing is Expensive
Because of the extensive testing in animals, routine safety pharmacology usually costs 1.0-1.5 million dollars/drug. Safety pharmacology is usually the major financial hurdle in the “Valley of Death” before an IND is issued and drug can be given to humans. Unexpected toxicity not apparent in animals can derail further drug testing. ~90% of drugs that begin human testing fail because of unexpected toxicity. As a result, strict attention to expensive safety pharmacology testing is needed before any IND is issued.
MNT drugs offer great promise to mimic the actions of endogenous NT proteins and DHEA, the parent molecule for MNT’s. By avoiding the sex steroid actions of DHEA and due to their increased potency at Trk receptors, MNT’s offer the benefits of survival-promoting NT actions on neurons. BNN27 in particular shows promise for helping motor neurons survive in ALS, which is primarily a disease of motor neuron death. MNT’s also hold great promise for other more prevalent brain diseases of aging, such as AD and PD. Enough preclinical work with BNN27 has now been completed. What is needed are the funds to carry out safety pharmacology testing so that an IND for BNN27 in ALS can be sought.