Metadoxine for fragile X---intermediate results

Alcobra just announced results from their trial of a proprietary formulation of metadoxine in fragile X subjects, and there is a glimmer of hope in their announcement (see here). While the drug did not show any beneficial effect on the primary outcome measure (essentially, an ADHD scale) it did result in some statistically significant improvement on 2 of 5 secondary measures. One of those showing improvement, the Vineland Adaptive Behavior Scale (VABS), is a well known and well validated outcome measure which has been used in other fragile X trials (though none of the other drugs resulted in improvement.) The other outcome measure showing improvement was the KiTAP, a computerized test of attention and distractibility. In both cases, the improvement was not only statistically significant, but also likely clinically meaningful; in other words, it was a relatively large effect.

The folks at Alcobra rightly note that this is the best showing to date for any drug in fragile X, and that both the KiTAP and VABS are appropriate and relevant outcome measures, and either could have been designated the primary outcome measure. The scale which was chosen as the primary endpoint, the ADHD RS-IV Inattentive subscale, was apparently chosen because the drug is thought to work mainly by enhancing attention, but the precise effect in fragile X could not have been anticipated.

It is possible that the VABS and KiTAP are just better or more useful scales for fragile X research, but it is also worth remembering that sometimes one or two secondary measures show apparent improvement which can’t be reproduced in subsequent trials. Alcobra has expressed interest in pursuing these findings, and they will presumably feature the VABS and KiTAP more prominently in the next round of metadoxine trials for fragile X. If they are able to reproduce these results, then this could be an important new treatment for fragile X.

Re-examining the nature of fragile X

In the wake of negative results from several high-profile clinical trials in fragile X, we find ourselves questioning all of our previous assumptions about the nature of this disorder. After all, understanding the basic pathology of the disease is critical to development of new treatments---this is true across the board, in all branches of medicine. In the early days, shortly after the FMR1 gene was discovered and the normal protein product of the gene (FMRP) was identified, it was noted that FMRP was an RNA binding protein. So, whatever the normal function of the missing protein was, it seemed to have something to do with RNA metabolism. Since RNA is the template that is used to make new proteins, this meant that the fragile X protein was involved in regulating protein synthesis. A bit more work over the next few years led to the “mGluR Theory” of fragile X, the idea the FMRP normally regulates protein synthesis in dendrites in response to synaptic activity, and that in the absence of FMRP (in fragile X) there was abnormal protein synthesis, leading to excessive activity in some signaling pathways.

While this theory was extensively validated, pointing to the potential therapeutic value of mGluR5 antagonists as treatments for fragile X, there was always the possibility that this represented only a piece of the puzzle. After all, most biomedical research is done in fairly narrowly-focused model systems; the whole point of using models (animals, cells, etc.) is to simplify the incredibly and incomprehensibly complex human brain to the point where experimentation is feasible. Thousands of experiments by hundreds of scientists around the world confirmed that absence of FMRP led to consistent signaling abnormalities, and that these could be normalized by decreasing the function of metabotropic glutamate receptors, either genetically or pharmacologically. So, the mGluR Theory isn’t necessarily wrong, but clearly there’s more to the story.

Keep in mind that all of the above takes place in the postsynaptic compartment, otherwise known as the dendrite, or the dendritic spine to be more specific. However, it has been known all along that FMRP is normally present in the presynaptic compartment, otherwise known as the axon, as well as in surrounding glial cells like astrocytes (these are the non-neurons in the brain that perform many of the household chores.) So, the question has always been, what’s FMRP doing in all those other places? There’s no protein synthesis in axons, and there’s no synaptic activity in glia, so FMRP must be doing more than just regulating activity-dependent protein synthesis.

In the years since the original conception of the mGluR Theory, some valuable clues have come along. For example, FRAXA scientist Dr. Len Kaczmarek first found that FMRP interacts directly with certain ion channels to regulate neuronal excitability; specifically, he reported that one section of FMRP (not the RNA binding part) interacts with a large potassium channel called Slack to fine-tune the fidelity of auditory circuits. More recently, another FRAXA researcher, Dr. Vitaly Klyachko found that FMRP interacts directly with the presynaptic BK channel---literally the Big K (potassium) channel---to regulate neuronal excitability, and that absence of FMRP results directly in hyperexcitability of this system. In collaboration with Dr. Steve Warren, they examined samples from a patient with a rare mutation of FMR1. This patient has a point mutation in the gene, which results in a single amino acid change in the protein, which is produced in normal amounts. The patient has what is described as a “partial fragile X phenotype” of intellectual disability and seizure disorder, but without the physical features or typical behaviors of fragile X syndrome. Upon examining the protein produced from this mutation, Dr. Klyachko found that it had entirely normal RNA binding properties, resulting in normal activity-dependent protein synthesis and normal mGluR-LTD. The only problem caused by this single amino acid change was that this mutated FMRP couldn’t interact with BK channels, leading to increased excitability of the axon and greater neurotransmitter release. In other words, there is an entirely separate and very different presynaptic function for FMRP, not related to its RNA binding function in dendrites, and this represents another facet of fragile X, a distinct presynaptic phenotype unrelated to mGluR5.

It has long been known that FMRP is highly expressed in the non-neuronal cells of the brain (glia). Additionally, early experiments showed that normal neurons growing with fragile X glia demonstrated many of the same abnormalities we would expect to see in fragile X neurons. Conversely, growing fragile X neurons with normal glia reversed many of the usual structural abnormalities. Clearly, something is going on in fragile X glia, and that represents a potentially significant part of the disease mechanism. More recently, FRAXA researcher Dr. Yongjie Yang has shown that fragile X astrocytes (one main type of glial cell) have low levels of a key glutamate transporter, and this deficiency contributes to excessive glutamate levels in the synapse, resulting in hyperexcitability. This abnormality does not respond to mGluR5 antagonists; in fact, they may aggravate the problem. Thus, we have identified key glial phenotypes which appear to be independent of the other phenotypes described in neurons.

As we move forward, we may need to consider the contributions of these distinct presynaptic, postsynaptic, and glial phenotypes to the clinical presentation of fragile X. We may need to treat them separately for best overall effect, and the knowledge that different things are going on in different places in the brain may help us to develop combination drug strategies which can significantly alter the course of fragile X.

Pharmacogenomic Testing in Psychiatry

A fellow clinician asked me what I thought about "pharmacogenomic testing" services which are now being offered, and heavily promoted. I guess people think of me as some kind of expert on genetics in psychiatry (hardly!), and I've previously mentioned that I don't think these tests are worthwhile.

Basically, this is genetic testing only for variations in metabolism of drugs; there are no genetic tests for any of the usual psychiatric disorders, and no tests of this kind are on these panels (I've looked at specific companies that offer this, but I won't name any names here---just commenting on the state of the technology.) Inaccurate diagnosis is one of the main reasons people don't respond well to treatment, and this testing doesn't provide any guidance there. So, this is the first issue.

There also aren't any validated tests for subgroups of depression or bipolar or schizophrenia that can tell you which patients are most likely to respond to which drug class (ie TCA vs SSRI vs SNRI.) That's #2 (that's probably coming next, though, and there is a lot of research going on to identify biomarkers for drug response within dx.)

#3 is that side effects and therapeutic effects (that's #4) don't always correspond to plasma levels, and so they certainly don't correspond very well to variations in metabolism. But if we just look at the levels achieved at a given dose (pharmacokinetics), then we can see that there are variations in absorption, distribution, and even elimination of the drug that have nothing to do with genetics (but are related to lifestyle, diet, weight, age, etc.) An obvious example: smokers metabolize benzodiazepines at twice the rate of non-smokers, on average. So, you might have a genotype which implies slow metabolism of benzos, but if you smoke, that gets you right back to average. These tests don't take that into account (some of them might ask if the patient smokes, but that's not a genotype). An even simpler example: you like to have a glass of grapefruit juice every morning; this makes you a very low 3A4 metabolizer, regardless of your genotype. So, it's much more important to ask people about their diet and lifestyle than it is to test their cytochrome genes.

There's also a parallel pharmacodynamic effect (that would be #5 and #6): two people with the exact same drug level can have radically different side effects and therapeutic results. This could be purely subjective (for example, one person simply has a low threshold for discomfort) but it also could be related to the actual physiological sensitivity of the target (usually a receptor and signaling pathway, in the case of psych meds.) There are many genetic variations in neurotransmitter receptors which are not fully understood (and not tested in these panels), and there are many more variations in signaling pathways which determine the sensitivity of target pathways, as well as those off-target pathways which cause side effects.

Surprisingly few drugs show tight correlations between plasma levels and therapeutic effects; obviously, you need a certain amount in your bloodstream to do anything, but in cases where there is a tight correlation, you can usually test for the level directly (as with TCAs.) For drugs like SSRIs, plasma levels aren't especially informative, and so they aren't routinely available.

However, in my mind, the main reason why these tests aren't especially useful (#7) is that you still need to use good clinical practice---you need to start the drug at a relatively low (subtherapeutic) dose in most cases, then increase gradually, monitoring for side effects. In the case of very long half-life drugs like Prozac or Abilify, you might start at a full dose, but you're essentially tapering up by letting the drug accumulate. You still need to inform the patient about likely side effects to watch out for, and you still need to assess whether an adequate response has been achieved, rather than placebo (another main reason for treatment failure.)

There's an argument to be made that this kind of testing can help to avoid those (rare) serious adverse effects which occur in unusually sensitive individuals who are very slow metabolizers, though I think any decent clinician should be able to catch these right away. One might also argue that this kind of testing can help identify patients who will definitely need higher doses for best effect (rapid metabolizers.) Here again, any competent clinician should know that the dose needs to be pushed because they aren't getting a real therapeutic effect, though admittedly very few clinicians seem to be capable of making the distinction between drug and placebo reliably. Still, you just can't assume that a patient who metabolizes a drug rapidly, and needs more of it for a true therapeutic effect, can actually tolerate the higher dose. That could be a major downside to having this kind of information, especially if less competent clinicians assume they need to simply start out at a high dose and push it up really quickly based on this kind of test.

In any case, it all comes down to cost. If this is cheap and easy, it could be useful in some cases, and post-marketing studies could make this kind of test even more useful. But, if it's expensive and time-consuming, I just don't think it's worth the trouble.

Something to be thankful for---a major success for Neuren

This isn’t a fragile X trial, but the same Neuren compound that is in trials now for fragile X (NNZ-2566) has shown significant positive effects in a Phase 2 trial for Rett syndrome.

The results of the trial are interesting, in that improvement was seen a Rett syndrome-specific rating scale compared to placebo, and there was also improvement noted on the CGI-I (Clinical Global Impression of Improvement) and Caregiver Top 3 Concerns. However, there was no effect seen on ABC scores (Aberrant Behavior Checklist) compared to placebo. Many in the fragile X field have noted the inadequacies of the ABC; indeed, it was never designed or intended to be an outcome measure for clinical trials. In this case, a Rett-specific rating scale called the Motor-Behavior Assessment (MBA) showed a statistically significant and clinically meaningful treatment effect at the highest dose of the Neuren compound compared to placebo.

This is great news for those of us in the fragile X field for several reasons. First of all, it shows that this compound really does something---it seems to have useful properties in actual patients, and that’s not trivial. Second of all, this result demonstrates that disease-specific symptoms can improve significantly on the drug, and that improvement can be measured in a relatively short clinical trial. Additionally, it shows that a drug can have beneficial effects on core features of a genetically based developmental disorder, even if the more general rating scales (like the ABC) show no change. This is strongly reminiscent of the experience of many families and clinicians in recent fragile X clinical trials, where the drugs showed no advantage compared to placebo, but genuine improvement was noted in many subjects, with significant deterioration upon discontinuation of the drugs. Thus the calls for improved rating scales which can “capture” these core, disease-specific therapeutic effects. The Neuren fragile X trial is using some fragile X-specific outcome measures which will hopefully lead to similar positive results. The fact that this result is good news for Neuren also means that the company should remain financially viable for longer, so that they can continue the development of this compound for a number of indications---more “shots on goal”.

Of course, the usual caveats apply: this was a small study, and these results need to be replicated in a larger Phase 3 trial. Still, there’s a realistic possibility that we may see a similar result in fragile X!

Here's a question we get all the time: I was wondering if the protein needed can be delivered another way, gene therapy, or via bacteria perhaps?

Many people who are new to fragile X ask us about protein replacement strategies of one kind or another; it's an excellent question, and it's one we've been thinking about for a long time. After all, people with fragile X are only missing a single protein---isn't the simplest and most effective possible treament to just put that protein back in there? When FRAXA first started funding biomedical research, we were especially interested in exploring the possiblities of protein replacement therapy because this type of treatment was being commercialized by several biotech companies for several inborn errors of metabolism, like Gaucher's Disease.

However, we've subsequently found that there are several problems unique to fragile X that make a protein replacement approach very difficult, perhaps impossible. It all has to do with the nature of FMRP itself---this protein is a key regulator of dendritic protein synthesis whose own translation is very tightly regulated, bother spatially (where) and temporally (when). We have tried to add the protein into cells in a number of ways, and it is always quite toxic, because the protein needs to be at very specific places, only at very specific times---putting FMRP into cells in any way that does not involve natural regulatory mechanisms is not good for those cells, and it certainly doesn't fix fragile X.

One way to add some natural regulation to the process of restoring FMRP to cells is through gene therapy---add a copy of the gene, and let the cell make its own FMRP. This can actually be done in vitro, and even in the mouse model, and it works---sort of. The problem is that the version of the gene added to cells in this manner isn't exactly the same; it typically is packaged in some kind of viral "vector" and has some kind of viral promoter to get the gene going. This produces FMRP, but not the same way as it's naturally produced. However, this is a relatively minor problem. The major problem with gene therapy technology right now is that it still doesn't allow for delivery to the whole brain. The gene therapy experiments that have been done in fragile X have been done by injecting the vector directly into small areas of the (very small) mouse brain with a needle; this is completely impractical in humans.

We have been waiting for a technical advance that would allow delivery of gene therapy vectors (viral or synthetic) globally, because we know this is necessary for fragile X. The whole brain is affected, not just one small area (even conditions like Parkinson's Disease which involve relatively small areas of the brain still can't be treated by gene therapy.) Once the technology advances to the point that life-threatening conditions like Tay-Sachs can be treated by global CNS gene therapy, we should be able to adapt those techniques to gene therapy for fragile X. For now, we're still waiting.

Lastly, the idea of gene re-activation is especially attractive in fragile X. Most people with fragile X have a trinucleotide repeat expansion in the non-coding promoter region of the gene---this means that their mutation leaves them with their genetic code intact to potentially produce perfectly good FMRP. The only problem is that the gene is transcriptionally silenced. This means that it is densely methylated and wound around packing proteins called histones, which are de-acetylated to stabilize the DNA in a compacted form. There are other mechanisms of gene silencing and regulation of transcription that we are only now learning about, so turning a gene back on is far from simple. Turning only one gene on in any kind of targeted fashion is entirely impossible at this time---the technology simply doesn't exist, though lots of scientists are working on this.

We frequently get simplistic proposals to use chemical demethylating agents and histone deacetylase inhibitors as a way to get people with fragile X to start making FMRP again. This is another one of those things that looks easy if you're studying cells in a dish, but it's much, much harder to do in a whole brain. It's potentially very dangerous, because any chemical that can reactivate FMR1 will almost certainly reactivate many other genes. Furthermore, at this time there is no way to test any reactivation strategies, because we don't have any animal models with a transcriptionally silenced trinucleotide repeat expansion. Many attempts have been made to construct a "knock-in" (KI) mouse, but no matter how big the section of CGG repeats scientists introduce, the KI mice don't silence the gene the way people do. So, we have knockin mice that are excellent models of FXTAS, but they don't have fragile X. This is a problem we've been attacking by funding development of mice with human fragile X neural stem cells grafted into their brains, but it's been slow going. Until something like that is available, reactivation strategies are dead in the water.

Can eating broccoli cure autism (or fragile X)?

A fascinating new study is getting a lot of attention lately, so I decided to give it a closer read. A group of MGH, Harvard, and UMass autism researchers tested a broccoli sprout extract containing the antioxidant and apparent active ingredient sulforaphane in a double-blind, placebo controlled trial (read the full text here).

The first caveat regarding this study is that the number of subjects is quite small; indeed, there were only 14 completers in the placebo group (using a 2:1 drug:placebo ratio), which could give rise to all kinds of misleading things. One thing I noticed was that there was no appreciable placebo response. An excessive placebo response can doom a trial, but when you see no placebo response at all, a red flag should go up in your mind. In small studies like this, the superiority of drug over placebo can result from an unusually small placebo response---a statistical fluke in the randomization process, really. If we then compare the placebo group showing no response to the drug group showing a typical placebo effect, but no actual treatment effect, it can appear that there is a big difference. This is essentially what happened in the first Novartis trial; the seven fully methylated subjects just happened to show no placebo response at all; in the larger study of the drug, this effect disappeared (in fact, the fully methylated group had an extra-large placebo effect.)

It was somewhat reassuring to see that ABC scores in the treatment group increased significantly 4 weeks after discontinuing, while still blind to treatment status, though there were numerous dropouts at this point, complicating interpretation. This kind of on/off effect is what you like to see, and is generally indicative of a true drug effect (of course it also means there is little carry-over effect, but most drugs do stop working when you stop taking them!) The effect of the broccoli sprout extract was significant, resulting in a 20+ point drop in ABC scores (note that the outcome measures were all the same as those used in recent fragile X trials.) However, the placebo response in the Novartis Phase IIb/III fragile X trials was actually quite similar in magnitude, so this could all be a statistical quirk.

Nonetheless, this is an intriguing result, especially since broccoli sprout extracts are widely available as nutritional supplements. But what about dosage? Can you actually get the same stuff used in this trial, and how much would you need? The study drug was a custom preparation which is not available anywhere, and the amount of sulforaphane given each day in the trial would be the equivalent of 20 or more capsules of the commercially available broccoli sprout extract, by my calculations. In addition, the potency of the extract was carefully monitored and maintained, implying that the compound is not entirely stable, and the pills you get at GNC might not even be as potent as they say (always an issue with unregulated nutritional supplements.) Still, 20 pills a day is possible as a treatment strategy. At this point, the evidence seems a bit weak, so I'd recommend waiting before trying this, but keep an eye on the broccoli story!

More bad news...but then some good news for fragile X research

Let's get the bad news out of the way first---Roche announced that the clinical trial of its lead mGluR5 antagonist for fragile X had failed to show any superiority on any of the outcome measures used (either primary or secondary.) They have also announced that they are cancelling their fragile X program. There aren't many details available at this point, though Roche has pledged to present and publish the data...eventually. I'm heading to the international mGluR conference in Sicily in a couple of days, and this is sure to be the hot topic; perhaps some new tidbits will be forthcoming. In any case, this was hardly a surprise. The Novartis results strongly suggested that the Roche compound would follow the same path; if anything, reports from the families participating in the Roche trial were even less promising than in the Novartis trial. If we are thinking that tolerance is a major problem, then the more potent and longer acting Roche drug may have even more problems with tolerance.

Now for some good news: the awards for the new fragile X research centers have been announced by the NIH. Three "Centers for Collaborative Research in Fragile X" will receive $35 million in funding over the next 5 years, and we couldn't be happier with the choices. These are all research groups that have been heavily supported by FRAXA over the years, and we think they will make a real difference, especially now that they have the resources to get things done!