a life in a lab
The recently released information about the toxic effect of the FAAH inhibitor BIA-10-2474 clearly indicates that there were no symptoms of toxicity observed up to 20 mg single dose in all subjects. The first symptoms of toxicity were detected when the the subjects received the next dose of 50 mg. It remains unclear how long the substance remains in the tissue and whether accumulation might have contributed to the toxicity. However, since they waited 19 days between the last cohort dosage and the next, this is unlikely. Therefore, a single dose of 50 mg of this compound is neurotoxic, again pointing towards non-FAAH mediated adverse effects.
For the sake of transparency in the ongoing discussion about why it was possible that BIA-10-2474 (the in the protocol mentioned substance 3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide) went into phase O/1 clinical trials one needs to critically study and understand the study protocol approved by the ANSM, French Ministry of Health. Interestingly and worryingly, there is no mention of "covalent" or "mechanism-based" inhibition, despite of the fact that the compound does exactly that. The study protocols even mentions the term "reversible FAAH" inhibitor, which is rather misleading without the biochemical context of the measurements that led to this conclusion. There are of course covalent reversible inhibitors, but reversibility in the physiological context may be different from in vitro assays. Moreover, how was specificity assessed and how was the mecahnism based action discussed in the context of safety?
The study protocol raises many questions: The study conduct enables high flexibility for the trial (decision making committee). maybe at the expense of safety for the people involved in the trial. In the introduction there is no mention about mechanism of action, potential for side effects on about 200 protein targets with an idential enzyme mechanism as FAAH. Also, it is somewhat unclear how fast dosing could be escalated. The stopping rule for escalation to next level “drug related severe AEs of the same character in 4 or more subjects” was rather undefined and apparently there was no stopping rule for serious adverse events.
I upload the study protocol that used to be confidential prior to the accident, but now in the public domain as it has been affecting people, researchers, drug developing companies and authorities.
This is a black Friday for Pharma drug development in general and for endocannabinoid research in particular. One would expect that the extensive preclinical development with small molecules ensures the lack of severe toxicity in humans once the experimental drug is taken into the develomental phase I in humans. This was not the case with the irreversible (covalent) FAAH inhibitor BIA-10-2474 from Bial, which was tested by Laboratoire Biotrial in Rennes, France. As Reuters reported "French drug trial disaster leaves one brain dead, five injured". Apparently the high chronic doses caused this effect in some humans. The question now is whether this toxicity is due to the mechanism of action (via inhibiting the degradation of endocannabinoids in the brain), or whether this is a severe aversive effect specific to the drug developed by Bial? Given that Pfizer already entered phase II clinical trials with the same or similar concept (potent, irreversible and selective FAAH inhibitor PF-04457845) and also Merck (MK-4409), the disaster may rather have been caused by the toxicity of this particular drug from Bial. Yet, how selective is BIA-10-2445 towards FAAH over other enzymes at higher concentrations? More information about the full spectrum of pharmacological effects of BIA-10-2472 is awaited and will have to be carefully analyzed by the research community. Furthermore, formation of haptens with serine residues in certain proteins like hydrolases (lipases, proteases or esterases) which in humans may lead to a strong and exacerbated immune reaction cannot be excluded without more detailed information about the cause of damage and lethality.
FAAH inhibition, leading to dramatically increased levels of the endocannabinoid anandamide and other N-acylethanolamines, may be a feasible novel therapeutic option to treat certain types of neuropsychiatric disorders and maybe certain forms of pain, but irreversible blockage of FAAH may likely also cause severe side effects in the kidney and liver, especially upon prolonged treatment. The fate of the covalent irreversible FAAH inhibitors for clinical development just got another blow by this sad report from Rennes, despite the fact that the toxic effect of BIA-10-2445 may be unrelated to the effect on the endocannabinoid system.
The basic biological phenomena rely on molecular interactions which are mostly non-covalent. When it comes to providing evidence that two molecules interact with each other, say e.g. two proteins that might communicate via binding to each other or a ligand and a receptor, an inhibitor and its target, etc. then we usually measure a dissociation constant. a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex falls apart into its component molecules, or when a salt splits up into its component ions.
The dissociation constant is commonly used to describe the affinity between a ligand (such as a drug or substrate) and a protein i.e. how tightly a ligand binds to a particular protein. Ligand-protein affinities are influenced by non-covalent intermolecular interactions between the two molecules such as hydrogen bonding, electrostatic and hydrophobic interactions and Van der Walls forces.
Reading manuscripts and published papers, there are cases where binding is used as an argument or proof, but often completely neglecting the underlying dissociation constant. E.g., when it comes to protein-protein interactions proteins are over-expressed and then co-immunoprecipitated, often without any control over the copy numbers of these proteins. Since binding is a function of concentration, anything will bind to anything when the concentration is high enough. The other case is that apparently specific inhibitors are used at such high concentrations (exceeding the dissociation constant originally measured) to prove that a phenomenon is mediated via a particular pathway or receptor. It is surprising how little concentration is taken into account! If you want to see what you want to see it does not seem to be a crime to rise the inhibitor concentration such that you finally see it. In order words, you have 50% chance that your inhibitor blocks your pathway and by rising its concentration, the chance will increase. To my opinion, many small organic molecules will inhibit a protein when its concentration is high enough. That is exactly why we need nanomolar inhibitors and also apply them at nanomolar conentrations.
Open access is a great model but as soon as it was introduced, it became a market model for preditors. As a consequence, the gold (author pays) open-access model has given rise to a great many new online publishers. Many of these publishers are not only spaming our inboxes, but are also corrupt and exist only to make money off the author processing charges that are billed to authors upon acceptance of their scientific manuscripts. For publicaitons, it may be better to be conservative and not waste time with an inflational publication strategy. Here is a useful list of publishers and journals that may not be clean:
The canonical triterpene amyrin was recently suggested to bind to CB1 receptors and to significantly mediate cannabimimetic effects in animal models of pain (Pain, 2011 Aug;152(8):1872-87). Andrea Chicca and Janine Marazzi in my lab now show that this molecule does NOT directly interact with CB receptors but inhibits the breakdown of the major endocannabinoid 2-AG. Thus, it is an indirect ligand at CB receptors, which may explain, at least in part, its analgesic and antiinflammatory effects. Interestingly, amyrin weakly targets both MAGL and ABHD hydrolases (partial inhibition) but the effect of this on 2-AG levels in brain tissue is superadditive. Since amyrin is a major constituent of many antiinflammatory and analgesic medicinal plants, we speculate that its presence is fundamentally involved in their pharmacological action.
Cells are made of proteins (among other things) and they interact with each other and change their properties. Proteins thus comunicate directly or indirectly and this communication is also called signalling (derived from Shannons theory of communication).
A good example are the MAP kinases, which interplay with each other via multiple phorsphorylation and dephosphorylation steps. In vitro this type of signalling can be studied relatively easily these days using gene and protein arrays. Today, whole journals are dedicated to cell signalling and thousands of papers dedicate their time to elucidate which signal leads to which signal and which one is a master switch in what context and so forth. I admit that when whole networks are studied this is very nice and has a big value on its own. A nice example is a recent paper on the self-organization and regulation of intrinsically disordered proteins with folded N-termini.
Experience in my lab shows that working with different cells you can get fantastic data (reproducible) one day and totally different data another day (again reproducible). For students this is very worrying and for me it's both worrying and interesting. It seems that these differences depend on the cell culture conditions and that there are close to chaotic processes involved, which basically make certain studies in cell signalling an art. Imagine that you need a complementary dataset for another experiment and you have two opposing datasets from signalling – would you chose the one that fits your wishful thinking? Or would you say that the data are strangely chaotic and beyond interpretation (or bullshit)? I just wonder whether a cell line in my lab ist he same cell line in your lab (at the level of signalling). You hear a critical undertone.
Maybe it’s the art of interpretation and the art of making big statements about something that is not much more than an in vitro artifact? This sounds like „Tamtam regulates Sharky which is a downstream modulator of Gugu – implications for cancer“ … just to give an fictive example. But what if in my lab Tamtam does not do anything and my antibody does not even recognize Gugu ? When I see (or review) studies in which artificially high concentrations of a pharmacological agent are studied at the level of signalling I always wonder whether this is real or not. Are the changes in gene expression or phosphorylation patterns in vitro really meaningful (robust or not robust)? It is our duty to do many experiments to find out. Imagine if your cells or a cell line from you tumour (in case you had cancer) would be useful to improve treatment of diseases. Unfortunately, the high hopes for a personalized medicine based on signalling (in vitro) was already kind of shattered by the discovery of the fraud of papers in New England Journal of Medicine and Nature Medicine – signalling is a difficult business, a ghost. If you want to get to know it (the gost) have a look at this one …….. (my personal favourite on Cellular Signalling)!
I just found this fantastic blog here called PRACTICAL FRAGMENTS by Dan Erlanson and Teddy Zartler. If you are interested in learning more about the pitfalls of drug discovery and development and you want to get a more global picture, this blog will certainly be of interest to you.
I remember those times when spam (unsolicited bulk emails) was limited to product selling, ranging from titanium fake watches, rather obscure agents to increase strength or health, free loans and morgages from Nigeria, cheap but nonetheless original Dior perfumes, designer bags, new parnters, and unnecessary software or even research antibodies. It was relatively easy to delete or block those from your inbox and declare it as spam. More recently it is the invitations to be a member in the editorial board of obscure new scientific journals, to participate in the editing of a book, or to publish an article in the WORLD journal of international biochemicophysicalneuroimmunologicalecologicalstructuralbiology, whatsoever. In India alone apparently every week one new online journal is born. At the same time, one new store of research products (they buy them cheap and sell them expensive) is openend. At universities we have obviously become the target of a new kind of spam!
The worst of all however are the invitations from BITs or ICDDT on "hot topics" of all sorts of research topics - usually they have 30% of all Nobel laureates (irrespective of their achievements) already invited and then lure you to participate in an event that is utterly unnecessary and most likely will NEVER take place (I wonder). Such mails are always declared as "conference invitation" with limited space. Invitation to my opinion ïmplies that you are invited. Over the last 2 years I must have received more than 30 such invitations from Chinese and Near East locations (Dubai seems to be a favorite place). There are also countless World congresses on drug discovery, molecular biology, ecology and genetics. Among many other such invitations, I have e.g. been invited to chair sessions at the 1st Annual World Congress of Molecular & Cell Biology. For only 2500 USD you can participate "for free" in this world-class event and of course, one should not miss it. Also, it is a great honor to be invited to such an event, isn't it? Thinking about it, could it be possible that such events have become a business oportunity for organizers to make business? Just yesterday I got an invitation to chair a whole session at the the 1st Annual World Congress of Marine Biotechnology. This is great, only that I don't work in this area, but that is a detail. Just out of curiosity I traced back one of the already passed "mega-congresses" in winter 2011, called "International Conference on Drug Discovery in Preclinical Research" which took place in Dubai in February 7-10th 2011. You will not be surprised to hear that that conference never took place. Now I face a dilemma, if I declare all conference invitations as spam, will I still be invited to the real ones? Time will show ...
I was stunned by a recent image obtained in our fluorescent microscope staining discrete parts of the cellular surface of membranes and wondered (and calculated) how many molecules are needed to make a visible staining. the molecule I used was approximately 20 Angström (2 nm) long - the surface of the cell maybe 100 microm2 and the number of molecules was 602000000000000. Then I saw a picture taken from space facing the earth illuminated by road lighting (see image from Nasa). How many hundreds of thousands of street lights are needed to make a road visible from approx. 350 km above the earth surface - not so many as it appears. BTW, the lense of the camera is about at the same relative distance as the lense of the microscope from the cell surface (only that the NASA picture is less zoomed in).
Nasa image showing road illumination and in orange the India-Pakistan boarderline