Ask Drugmonkey: Call to the Hivemind on Behavioral Neuroscience coursework

A longtime Reader asks:

My colleagues and I are trying to finalize our revisions/updates to the courses we will require as part of a PhD in behavioral neuroscience. It would be helpful to get input on what others’ experience is: how many credit hours of classwork are required, and what are seen as the essential items? [We’re at 47 class credits currently, trying to reduce to either 41 or 38 but facing resistance to eliminating non-neuro psychology classes from requirements.]

Anyone have any thoughts on this?

I myself think that “eliminating non-neuro psychology classes” is a huge mistake and I join their local resistance. The field of so-called behavioral neuroscience already has far too many people who are insufficiently grounded in good old Behavioral Psychology.

If you take the current replication hoopla seriously, it is a bad idea to cut behavior out of the curriculum.

Nature publishes overwhelmingly proven “NEW AMAZING FINDING” ….because optogenetics!

The PR headlines are breathless and consistent:

Researchers Identify Brain Circuit That Regulates Thirst

Brain’s On-Off Thirst Switch Identified

The paper is here.

Yuki Oka, Mingyu Ye & Charles S. Zuker Thirst driving and suppressing signals encoded by distinct neural populations in the brain Nature (2015) doi:10.1038/nature14108

The takeaway punch message from the Abstract:

These results reveal an innate brain circuit that can turn an animal’s water-drinking behaviour on and off, and probably functions as a centre for thirst control in the
mammalian brain.

Somebody like me immediately thinks to himself “subfornical neurons control drinking behavior? This is like the fifth lecture in Psych 105: Introduction to Physiological Psychology.”

Let’s do a little PubMed troll for “subfornical drinking“. Yeah, we’ve known since at least the 1970s that the subfornical control of drinking behavior is essential, robust and mediated by angiotensin II signalling. We know how this area responds to blood volemia and natremia and how the positioning relative to the third ventricle and the function of the circumventricular organ vis a vis the blood-brain barrier permits this rapid-response. We know the signalling works through AT1 receptor subtype to excite subfornical neuronal activity via electrophysiological recording techniques and genetic deletions. Cholinergic mechanisms have likewise been identified as critical components via pharmacological experiments. Mapping of activated neurons has been used to identify related circuitry. The targets of subfornical neurons are known and their involvement in drinking behavior has likewise been characterized. Extensively. We know that electrical stimulation of these neuronal populations activates drinking in water sated rats, for goodness sake! We know there are at least three subpopulations of SFO neurons involved and something about the neurochemical signalling complexity.

There are review articles that you can read if you want to get up to speed.

The new work by Oka and colleagues simply repeats the above-mentioned electro-stimulation experiment from 1983 using optogenetic stimulation. Apart from this, maybe, we have an advance* in that they identified ETV-1 vs VGAT (GABA transporter) markers of two distinct subpopulations of neurons which have opposite effects on the motivation to consume water.

That’s it.

This paper is best described as a very small, incremental advance in understanding of thirst and drinking behavior, albeit tarted up with the pizzaz of optogenetic techniques.

Yet it was published in Nature.

Someone really needs to introduce the editorial staff of Nature to PubMed.
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*BTW, a Nature editor confirms this microscopic incremental advance is what is new about this paper.

Failure to Replicate

I should have put that in quotes because it actually appears in the title of this new paper published in Neuropsychopharmacology:

Hart AB, de Wit H, Palmer AA. Candidate gene studies of a promising intermediate phenotype: failure to replicate. Neuropsychopharmacology. 2013 Apr;38(5):802-16. doi: 10.1038/npp.2012.245. Epub 2012 Dec 3. [PubMed]

from the Abstract alone you can get a sense

We previously conducted a series of 12 candidate gene analyses of acute subjective and physiological responses to amphetamine in 99-162 healthy human volunteers (ADORA2A, SLC6A3, BDNF, SLC6A4, CSNK1E, SLC6A2, DRD2, FAAH, COMT, OPRM1). Here, we report our attempt to replicate these findings in over 200 additional participants ascertained using identical methodology. We were unable to replicate any of our previous findings.

The team, with de Wit’s lab expert on the human phenotyping and drug-response side and Palmer’s lab expert on the genetics, has been after genetic differences that mediate differential response to amphetamine for some time. There’s a human end and a mouse end to the overall program which has been fairly prolific.

In terms of human results, they have previously reported effects as varied as:
-association of an adenosine receptor gene polymorphism with degree of anxiety in response to amphetamine
-association of a dopamine transporter gene promotor polymorphism with feeling the drug effect and diastolic blood pressure
-association of casein-kinase I epsilon gene polymophisms with feeling the drug effect
-association with fatty acid amide hydrolase (FAAH) with Arousal and Fatigue responses to amphetamine
-association of mu 1 opioid receptor gene polymorphisms with Amphetamine scale subjective report in response to amphetamine

There were a dozen in total and for the most part the replication attempt with a new group of subjects failed to confirm the prior observation. The Discussion is almost plaintive at the start:

This study is striking because we were attempting to replicate apparently robust findings related to well-studied candidate genes. We used a relatively large number of new participants for the replication, and their data were collected and analyzed using identical procedures. Thus, our study did not suffer from the heterogeneity in phenotyping procedures implicated in previous failures to replicate other candidate gene studies (Ho et al, 2010; Mathieson et al, 2012). The failure of our associations to replicate suggests that most or all of our original results were false positives.

The authors then go on to discuss a number of obvious issues that may have led to the prior “false positives”.

-variation in the ethnic makeup of various samples- one reanalysis using ancestry as covariate didn’t change their prior results.

-power in Genome-Wide association studies is low because effect sizes / contribution to variance by rare alleles is small. they point out that candidate gene studies continue to report large effect sizes that are probably very unlikely in the broad scheme of things…and therefore comparatively likely to be false positives.

-multiple comparisons. They point out that not even all of their prior papers applied multiple comparisons corrections against the inflation of alpha (the false positive rate, in essence) and certainly they did no such thing for the 12 findings that were reported in a number of independent publications. As they note, the adjusted p value for the “322 primary tests performed in this study” (i.e., the same number included in the several papers which they were trying to replicate) would be 0.00015.

-publication bias. This discussion covers the usual (ignoring all the negative outcomes) but the interesting thing is the confession on something many of us (yes me) do that isn’t really addressed in the formal correction procedures for multiple comparisons.

Similarly, we sometimes considered several alternative methods for calculating phenotypes (eg, peak change score summarization vs area under the curve, which tend to be highly but incompletely correlated). It seems very likely that the candidate gene literature frequently reflects this sort of publication bias, which represents a special case of uncorrected multiple testing.

This is a fascinating read. The authors make no bones about the fact that they’ve found that no less than 12 papers that they have published were the result of false positives. Not wrong…not fraudulent. Let us be clear. We must assume they were published with peer review, analysis techniques and samples sizes that were (and are?) standard for the field.

But they are not true.

The authors offer up solutions of larger sample sizes, better corrections for multiple comparisons and a need for replication. Of these, the last one seems the best and most likely solution. Like it or not, research funding is limited and there will always be a sliding scale. At first we have pilot experiments or even anecdotal observations to put us on the track. We do one study, limited by the available resources. Positive outcomes justify throwing more resources at the question. Interesting findings can stimulate other labs to join the party. Over time, the essential features of the original observation or finding are either confirmed or consigned to the bin of “likely false alarm”.

This is how science progresses. So while we can use experiences like this to define what is a target sample size and scope for a real experiment, I’m not sure that we can ever overcome the problems of publication bias and cherry picking results from amongst multiple analyses of a dataset. At first, anyway. The way to overcome it is for the lab or field to hold a result in mind as tentatively true and then proceed to replicate it in different ways.

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UPDATE: I originally forgot to put in my standard disclaimer that I’m professionally acquainted with one or more of the authors of this work.

Hart, A., de Wit, H., & Palmer, A. (2012). Candidate Gene Studies of a Promising Intermediate Phenotype: Failure to Replicate Neuropsychopharmacology, 38 (5), 802-816 DOI: 10.1038/npp.2012.245

Effects of MDPV ("Bath Salts") interact with the ambient temperature

A new paper from the Fantegrossi laboratory examines the behavioral and physiological effects of the substituted cathinone drug, and “bath salts” constituent, 3,4-methylenedioxypyrovalerone (MDPV) [ Search PubMed ] which is the compound which has dominated the US media reports of averse consequences of bath salts intoxication. To the extent that verification of the drug has been provided in such reports, of course. Additional confirmation can be found here, here.

The current issue of Neuropsychopharmacology has a bath salts image on the cover and contains an article from Baumann and colleagues on MDPV pharmacology (I discussed it here) and this paper from Fantegrossi and colleagues.

William E Fantegrossi, Brenda M Gannon, Sarah M Zimmerman and Kenner C Rice In vivo Effects of Abused ‘Bath Salt’ Constituent 3,4-methylenedioxypyrovalerone (MDPV) in Mice: Drug Discrimination, Thermoregulation, and Locomotor Activity. Neuropsychopharmacology (2013) 38, 563–573; doi:10.1038/npp.2012.233; published online 5 December 2012 [ ArticleLink(free); PDF ]

This is a behavioral pharmacology study in male NIH Swiss mice which first uses drug discrimination techniques to show that when mice are trained to discriminate 0.3 mg/kg i.p. MDPV from saline the subsequent dose response curves for 0.01 to 0.3 mg/kg of MDPV, METH and MDMA are nearly identical. This article has been made freely available so I won’t belabor this part of the study.

What I wanted to focus on was the radiotelemetry studies of body temperature and locomotion. For reasons related to this classic paper on MDMA from Malberg and Seiden, most investigations of the effects of stimulant drugs in rodents should include some consideration of the role of ambient temperature. Fantegrossi and colleagues examined the effects of 0.3-30 mg/kg i.p. MDPV at both 20°C and 28°C. They showed, first of all, that MDPV produces no change in body temperature when administered at 20°C, but induces temperature elevations in a dose-dependent manner when animals are evaluated at 28°C. Even more interesting is what is shown in Figure 4 which I’ve included here. You can see that the locomotor stimulant effect (total activity counts over 6 hrs; left panel) of MDPV also is more pronounced at the higher ambient temperature with a peak differential observed after the 10 mg/kg i.p. dose (timecourse for this dose shown in right panel). There were also some other interesting phenomenological differences observed with the high ambient temperature condition.

At the highest tested dose of MDPV (30 mg/kg), significant focused stereotypy was observed at 28 1C, but not at 20 1C. Furthermore, four (of six) mice treated with 30 mg/kg MDPV at the high ambient temperature engaged in skin-picking and self-biting, which drew blood, and, in accordance with our IACUC approval, were removed from the study and euthanized. No signs of self-injurious behavior were observed at any dose of MDPV administered at 20 1C.

Repetitive, stereotyped behavior is common with locomotor stimulants and can be observed following high doses of amphetamine, methamphetamine and cocaine among other compounds. So this is probably an expected effect. What was interesting here was the dependency on ambient temperature. Off the top of my head, I can’t remember either the stimulant drug sterotypy literature (which focuses on charcterizing the repetitive behaviors) or the locomotor studies (where the “inverted U” dose effect function often reflects the emergence of stereotyped behavior after high doses) focusing too heavily on the ambient temperature issue. No doubt I could stand to go back and review some papers with a closer eye on the ambient temperature.

This study, however, points a finger at environmental issues when trying to figure out the degree to which the drug MDPV might cause sensational media-friendly outcomes in some users. Studies such as the present one may indicate that factors as subtle as how hot it is the day a person takes a given drug can change the experience from relatively benign into something much more severe. Thus, a dose of a drug which has been taken before by the same user may have highly unpredictable effects just based on this one difference in the situation.

ADDITIONAL READING

Watterson et al 2012 demonstrated intravenous self-administration in rats.
Huang et al, 2012 showed locomotor effects of MDPV on activity wheels in rats.
Fuwa et al 2007 shows dopamine responses with microdialysis and locomotor effects [in Japanese, but the Abstract is in English and the figures are easily interpreted]
Meltzer et al 2006 present monoamine pharmacology on a series of pyrovalerone compounds

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Fantegrossi WE, Gannon BM, Zimmerman SM, & Rice KC (2012). In vivo Effects of Abused ‘Bath Salt’ Constituent 3,4-methylenedioxypyrovalerone (MDPV) in Mice: Drug Discrimination, Thermoregulation, and Locomotor Activity. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology PMID: 23212455

PSA: Beware the 'nip

ht: @merz @dirk57

Should NIMH be funding Me-Too drug development?

Hard on the heels of something I just learned about at a recent conference, the NIMH issued a Press Release for a new clinical trial they funded.

A drug that works through the same brain mechanism as the fast-acting antidepressant ketamine briefly improved treatment-resistant patients’ depression symptoms in minutes, with minimal untoward side effects, in a clinical trial conducted by the National Institutes of Health. The experimental agent, called AZD6765, acts through the brain’s glutamate chemical messenger system.

Interesting. The background is that prior studies* have shown that the dissociative anesthetic ketamine is capable of the rapid (within hours) amelioration of depressive symptoms. Yes, ketamine. The recreational drug known as Special K and the veterinary anesthetic they’ve used on your pet cat or dog. Same ketamine that is approved for human use in pediatric anesthesia, emergency medicine in some cases and for tricky clinical situations.

The same ketamine that has been widely used for decades in humans and nonhuman animals. It has established efficacy, mechanism of action and a huge therapeutic index. A big distance between effective doses and the dose that will kill you. Whether effect is recreational, medical or veterinary. Meaning it is safe.

So why are the studies (cited below*) of effect in depression so exciting? Because traditional drug therapy for depression takes weeks to have effect. Weeks of daily dosing. Selective Serotonin Reuptake Inhibitors (SSRIs) like Prozac are broadly familiar to most of my Readers, I would assume. Efficacy with these front-line meds takes up to three weeks to see effect on depressive symptoms. Trouble is, some cases of depression are acutely suicidal–they may just kill themselves before any SSRI has a chance to make them feel better. And hell, who wants to wait three weeks if another med could make you feel better by tomorrow? Prior to the ketamine work, the only other thing that seemed to have such a rapid effect was ECT. Yeah, ElectroConvulsive Therapy. Which has come a loooooong way from the One Flew Over the Cuckoo’s Nest era….but still. A single ketamine dosing seems quite preferable.

So…..on to the me-too drug development! Woot!
Zarate CA Jr, Mathews D, Ibrahim L, Chaves JF, Marquardt C, Ukoh I, Jolkovsky L, Brutsche NE, Smith MA, Luckenbaugh DA. A Randomized Trial of a Low-Trapping Nonselective N-Methyl-D-Aspartate Channel Blocker in Major Depression. Biol Psychiatry. 2012 Nov 30. pii: S0006-3223(12)00941-9. doi: 10.1016/j.biopsych.2012.10.019. [Epub ahead of print][Publisher, PubMed]

This AZD6765 compound is, as you might deduce from the letters, property of AstraZeneca Pharmaceuticals and indeed one of the authors lists this as his affiliation. The rest of the folks are from the NIMH intramural program which, presumably, provided the majority of the funding for the study.

The conclusions appear to be that this novel compounds, with a similar mechanism of action as ketamine worked but less well. From the Presser:

About 32 percent of 22 treatment-resistant depressed patients infused with ASD6765 showed a clinically meaningful antidepressant response at 80 minutes after infusion that lasted for about half an hour – with residual antidepressant effects lasting two days for some. By contrast, 52 percent of patients receiving ketamine show a comparable response, with effects still detectable at seven days. So a single infusion of ketamine produces more robust and sustained improvement, but most patients continue to experience some symptoms with both drugs.

However, depression rating scores were significantly better among patients who received AZD6765 than in those who received placebos. The researchers deemed this noteworthy, since, on average, these patients had failed to improve in seven past antidepressant trials, and nearly half failed to respond to electroconvulsive therapy (ECT).

So this is good. Anything that shows promise as a rapid-alleviator of depression is good by my lights.

But why is NIMH spending taxpayer dollars to develop me-too drugs? Look, I recognize that drugs within a class of pharmacological mechanism, like the SSRIs, may be differentially effective for different patients. And it is a good thing if we have more options to tailor medication to the individual patient. ADHD is another situation where an array of monoamine transporter inhibitors, including methylphenidate and amphetamine, are used with success and failure. One drug works for one patient, another works for a different patient….and they might describe the other medication as even worse than not being treated. So…great.

But make no mistake. The central feature driving me-too drug development is profit. Drug companies decide they can take a big enough slice of the market away from the market-leader to make it worth their while. Perhaps they had development in parallel and had sunk enough cost in by the time their competitor gained FDA approval that there was no turning back. Whatever. Point being that they are in it for the money and not for some noble cause of serving that subset of patients that do not gain relief from their competitor’s drug.

Over the past few years the side-chatter about the ketamine effect on depression has frequently been a lament about the lack of financial motive for companies to push forward with ketamine. Push forward with specific clinical trials to gain on-label approval for the indication of depression. Push forward with marketing campaigns. Push forward with physician education and stroking like they do with their proprietary stuff.

The Zarate paper took a stab at claiming the reason for developing something else was an attempt to avoid the adverse effects of ketamine. The dissociative type effects can be unpleasant and recovery doesn’t look fun. So there’s some toehold there to claim one is motivated to find a “perfect” drug which somehow produces the therapeutic effect with nothing else. Color me skeptical, given what I know about existing NMDA channel blockers like ketamine (and PCP, did I mention that? Yeah, angel dust might work for depression….).

So I smell profit motive in this effort.

What I don’t understand is why NIMH is involved with this. Why not just pursue the evidence body for ketamine?
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*References pulled out of the paper
R.M. Berman, A. Cappiello, A. Anand, D.A. Oren, G.R. Heninger, D.S. Charney et al. Antidepressant effects of ketamine in depressed patients. Biol Psychiatry, 47 (2000), pp. 351–354

N. Diazgranados, L. Ibrahim, N.E. Brutsche, A. Newberg, P. Kronstein, S. Khalife et al. A randomized add-on trial of an N-methyl-D-aspartate antagonist in treatment-resistant bipolar depression. Arch Gen Psychiatry, 67 (2010), pp. 793–802

C.A. Zarate Jr, N.E. Brutsche, L. Ibrahim, J. Franco-Chaves, N. Diazgranados, A. Cravchik et al. Replication of ketamine’s antidepressant efficacy in bipolar depression: A randomized controlled add-on trial Biol Psychiatry, 71 (2012), pp. 939–946

G.W. Valentine, G.F. Mason, R. Gomez, M. Fasula, J. Watzl, B. Pittman et al. The antidepressant effect of ketamine is not associated with changes in occipital amino acid neurotransmitter content as measured by [(1)H]-MRS Psychiatry Res, 191 (2011), pp. 122–127

M. aan het Rot, K.A. Collins, J.W. Murrough, A.M. Perez, D.L. Reich, D.S. Charney et al. Safety and efficacy of repeated-dose intravenous ketamine for treatment-resistant depression Biol Psychiatry, 67 (2010), pp. 139–145

Gaining clarity on the pharmacology of the bath salt MDPV

There are two new pharmacological investigations on the substituted cathinone drugs that have been discussed here on occasion.

Each of

Baumann MH, Partilla JS, Lehner KR, Thorndike EB, Hoffman AF, Holy M, Rothman RB, Goldberg SR, Lupica CR, Sitte HH, Brandt SD, Tella SR, Cozzi NV, Schindler CW.Powerful Cocaine-Like Actions of 3,4-Methylenedioxypyrovalerone (MDPV), a Principal Constituent of Psychoactive ‘Bath Salts’ Products. Neuropsychopharmacology. 2012 Oct 17. doi: 10.1038/npp.2012.204.

and

Simmler LD, Buser TA, Donzelli M, Schramm Y, Dieu LH, Huwyler J, Chaboz S, Hoener MC, Liechti ME.Pharmacological characterization of designer cathinones in vitro. Br J Pharmacol. 2012 Aug 17. doi: 10.1111/j.1476-5381.2012.02145.x.

report very similar findings for MDPV, the cathinone that appears most frequently in US newspaper reports.

As a very general rule, the amphetamine class stimulants do a couple of things to enhance the neuron-to-neuron chemical communication that occurs in the brain. The most common and significant effects tend to involve the transporter mechanisms that remove dopamine, norepinephrine and / or serotonin from the synapse, the gap between two neurons. These transporter molecules are an integral part of terminating a signalling event which has been caused by the release of one of these three monoamines from one of the neurons in question. Interfere with the operation of these transporters and a drug can potentiate the magnitude or duration of a given signalling event (i.e., release of one of the dopamine (DA), norepinephrine (NE) or serotonin neurotransmitters).

The amphetamine class stimulants have these properties. As does cocaine. As do therapeutic drugs such as methylphenidate (Ritalin) and Prozac. The term selective serotonin re-uptake inhibitor, SSRI, for Prozac-class antidepressant drugs refers to the transporter, obviously, and also indicates a key thing with the term “selective”. Drugs which have the ability to interact with one of the monoamine transporters tend to interact with the other ones as well. Substantial differences in effect can be associated with differences in the relative ability a specific molecule has to attach to the DAT versus the NET versus the SERotonin transporter (SERT). As one clear example, methamphetamine and MDMA differ in their relative ability to inhibit the SERT….this property of MDMA is associated with many of it’s stimulant-atypical properties relative to other amphetamine-class drugs.

The new studies both show that MDPV blocks all three transporters with much more potent effects at the DAT and NET relative to SERT. As Baumann and colleagues note, MDPV is 50 and 10 times more potent than cocaine (not an amphetamine, we’ll come to this) at DAT and NET respectively. Simmler and colleagues similarly indicate that MDPV is much more potent at DAT than cocaine or methamphetamine which did not qualitatively differ from each other.

So to this point, MDPV looks like a high-potency traditional stimulant. Most effective at the DAT, fairly effective at the NET and with less ability to block the SERT.

Cocaine and the amphetamines diverge at this point because the amphetamines act as a substrate at the transporters. Instead of only interfering and blocking them from doing anything, the amphetamines actually substitute for the neurotransmitter in question and are taken up into the cell. In so doing, they also cause an exchange to happen whereby the transporter moves some neurotransmitter from inside the cell back into the synapse. This transporter mediated efflux contributes to any “regular” release of neurotransmitter mediated through the merging of intracellular sacs (called vesicles) with the cell membrane.

The two papers agree in finding that MDPV has no ability to cause transporter-mediated efflux of dopamine and is therefore best categorized neuropharmacologically as a “pure” blocker (like cocaine) rather than an amphetamine-like transporter substrate.

The Simmler paper adds an in vitro model of blood/brain barrier penetration…in very simple terms the degree to which a molecule is fat-liking versus water-liking can alter the speed at which it can cross cell membranes and get into the brain. This paper used an in vitro preparation of human capillary endothelial cells (that form the blood-brain barrier) to show that MDPV is likely to cross the blood-brain barrier very rapidly, consistent with high lipophilicity predicted from its structure.

The upshot of the two papers is that MDPV shows pharmacological properties consistent with classical stimulants. It shows relatively high selectivity for DAT over SERT and high potency relative to drugs such as methamphetamine or cocaine. In vivo neurochemistry in the Baumann paper confirm that MDPV has potent effects on dopamine levels in the nucleus accumbens, a hallmark of drugs (beyond the stimulants even) which have substantial risk for compulsive use. The only somewhat discordant note for the structure-activity nerds is that MDPV looks so much like the rest of the amphetamines and cathinones that it will be interesting to discover why it doesn’t act as a transporter substrate (Simmler et al included a number of other cathinones and showed that many of them do act as transporter substrates.)

Together these papers suggest that MDPV has high abuse liability with a use pattern characterized by frequent re-dosing much like one sees with cocaine. This is consistent with many self-reports that are emerging from people who use MDPV and therefore, despite the relatively brief time on the “market”, we can predict a cocaine-like dependence problem to emerge for MDPV in the near future.

Single oral dose MDMA neurotoxicity revisited

I have been awaiting this paper since I saw the poster a few meetings back, Dear Reader. It contributes to an ongoing theme of posts on MDMA (Ecstasy) that I have neglected for some time. Some of you may recall the topic of my third blog post which noted the current attempts to get MDMA approved as adjunctive treatment during PTSD psychotherapy. I have been somewhat critical of their approach, mostly because of my understanding of the MDMA-associated neurotoxicity literature in animal species.

To overview, very briefly, if you administer MDMA twice per day at approximately 6-12 hr intervals for four days to rats, monkeys and a few other species, you produce lasting decrements in many markers for serotonin neurons / serotonin function in the brain. Lasting as in, as long as 7 years in a monkey (Hatzidimitriou, 1999). There is a parallel literature identifying lasting affective and cognitive alterations in human consumers of MDMA / Ecstasy and some imaging evidence of similar serotonergic changes.

It is tempting to associate at least the affective disruptions with the lasting serotonergic alterations. (Three monkey studies failed to connect these serotonergic patterns to substantial changes in cognitive behavior so that one is a little more tenuous…you can find some hints in the rat literature and some mother’s eye stuff in the monkey studies but the gun ain’t smoking very much.) However, as you are aware DearReader, the human studies of drug users are fraught with complications. One doesn’t know anything about pre-existing differences (depressives more likely to use MDMA?), the precise dose and pattern folks exposed themselves to, the environmental conditions, co-administered psychoactive substances and even the identity of the drugs being consumed as “Ecstasy”.

This cycles the discussion back to the controlled animal models. The MDMA enthusiast is frequently found to contest the relevance of the animal models, primarily on the grounds of the dose. The typical animal model features 10-20 mg/kg of MDMA per injection in rats and 5-10 mg/kg in monkeys. Again, these are repeated twice daily for four days. In addition, the route of administration is typically intraperitoneal (rat) and either intramuscular or subcutaneous in monkeys. Naturally, the majority of human use is oral which Pharm101 tells us should reduce the peak brain exposure as well as the rapidity with which peak levels are attained compared with the injected routes. So there has been vigorous debate, including between animal-research and human-research scientists, as to whether the animal data should be taken as relevant to the human condition.

As I blogged before, there is another concept from Pharm101 that relates to this discussion, i.e., that of species-scaling of drug doses. The short version is that you need higher per-kilogram-of-bodyweight doses in smaller species to produce similar outcome on parameters such as peak plasma levels, Area Under the Curve as well as toxic outcomes for various body systems including the brain. That prior post lays out data which show that a 1.6 mg/kg oral MDMA dose in a human produces peak plasma levels similar to 2.8 mg/kg in a squirrel monkey but an AUC similar to 5.7 mg/kg in a squirrel monkey (with a higher peak, obviously).

All well and good but the evidence of lasting serotonin changes on the low-end of the dosing spectrum has not been all that good. There was an old Ricaurte paper from the early days that found serotonergic changes in a handful of brain regions after a single oral dose of 5 mg/kg MDMA in squirrel monkeys. The trouble is, it was never replicated by any other papers and it was only in 3 subjects. So…not quite as convincing as the data on the higher-dose, injected, repeated models which come from multiple labs, in several species of laboratory animals and in many (total) animals per species.

A new paper from the Ricaurte group,

Mueller M, Yuan J, McCann UD, Hatzidimitriou G, Ricaurte GA. Single oral doses of (±) 3,4-methylenedioxymethamphetamine (‘Ecstasy’) produce lasting serotonergic deficits in non-human primates: relationship to plasma drug and metabolite concentrations. Int J Neuropsychopharmacol. 2012 Jul 24:1-11. [Epub ahead of print] [PubMed]

provides a long-past-due update on their prior report (Ricaurte et al, 1988; PubMed).

The study tested single oral doses of 5.7 (N=8), 10.0 (N=6) and 14.3 (N=4) mg/kg MDMA and the brains were collected one week later for analysis. As with prior studies, significant reductions in brain content of serotonin and its major metabolite 5-HIAA were observed in multiple brain regions including frontal, temporal, parietal and occipital cortex, the hippocampus, caudate nucleus, putamen and thalamus. Importantly, these reductions were dose-dependent in magnitude with some differences from the vehicle control group (N=8) failing to reach statistical significance. The lowest dose, however, did produce significant reductions of serotonin in frontal and temporal cortex, hippocampus and caudate.

This last is the most critical contribution because it replicates the prior study in a larger sample.

The one oddest thing about the design was the collection of brains at one week instead of two. For the vast majority of studies in this area, two weeks seems to be the modal time for brain harvest. I think the choice of one week here is going to muddy the waters because there will be those that claim this is reflective of acute depletion of serotonin stores rather than the classic neurotoxicity profile. Concerns are partially alleviated by some serotonin transporter binding data provided suggesting reduced expression, but only a single brain slice per treatment group was shown. It would have been nicer if this had been a completed study with quantification from all animals. They authors have left some daylight for their critics and it is not really clear why they would have done this.

In the discussion, the authors continue their thesis that 5.7 mg/kg is equivalent to 1.6 mg/kg for a human. Therefore, they conclude that they have shown that lasting serotonergic deficits can be produced at doses that are unarguable “typical human doses” of MDMA. I have previously argued that this is a dose range that is being used in the clinical protocols even if you leave off notions of species scaling. So overall, yes I would say I agree with their basic contention that they have shown the expected serotonergic effects with MDMA exposure that is 1) oral, 2) single-dose and 3) within the range of expected human single-use episodes.

This study should further convince those who have previously argued that the animal data has no relevance because of dosing issues. This shows that there is no magic threshold of protection that happens to coincide with notions of “typical human use”.

"Chemical Free" is bullshit. But you knew that already…..

Repost- Faces of Neuropsychopharmacology: Percy L. Julian, Ph.D.

Sorry folks, I’m swamped lately. Kept meaning to do something for Black History Month that was new but I haven’t managed to get to it. So I’ll repost this from a few years back. It originally appeared on the SB blog Feb 17, 2009.

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sourcePercy Lavon Julian, Ph.D. (1899-1975) was a scientist who rose from humble beginnings, was trained and educated in an adverse cultural era and became a highly accomplished synthetic chemist and entrepreneur (Wikipedia; PubMed; ACS bio). From the American Chemical Society biography:

He was born in Montgomery, Alabama, on April 11, 1899, the son of a railway clerk and the grandson of slaves. From the beginning, he did well in school, but there was no public high school for African-Americans in Montgomery. Julian graduated from an all-black normal school inadequately prepared for college. Even so, in the fall of 1916, at the age of 17, he was accepted as a subfreshman at DePauw University in Greencastle, Indiana. This meant that in addition to his regular college courses he took remedial classes at a nearby high school. He also had to work in order to pay his college expenses. Nevertheless, he excelled. Julian was elected to Phi Beta Kappa and graduated with a B.A. degree in 1920 as valedictorian of his class.

On discovering new medicines

A link from writedit pointed me to a review of drugs that were approved in the US with an eye to how they were identified. Swinney and Anthony (2011) identified 259 agents that were approved by the US FDA between 1999 and 2008. They then identified 75 which were “first in class”, i.e., not just me-too drugs or new formulations of existing drugs or whatnot. There were 20 imaging agents, not further discussed, and 164 “follower” drugs.

The review also focused mostly on small molecule drugs instead of “biologics” because of an assumption that the latter are all exclusively “target based” discoveries. The main interest was in determining if the remaining small molecule drugs were discovered the smart way or the dumb way. That’s my formulation of what the authors term “target based screening” (which may include “molecular mechanism of action”) discovery and “phenotypic screening” type of discovery. As they put it:

The strengths of the target-based approach include the ability to apply molecular and chemical knowledge to investigate specific molecular hypotheses, and the ability to apply both small-molecule screening strategies (which can often be achieved using high-throughput formats) and biologic-based approaches, such as identifying monoclonal antibodies. A disadvantage of the target-based approach is that the solution to the specific molecular hypotheses may not be relevant to the disease pathogenesis or provide a sufficient therapeutic index.

A strength of the phenotypic approach is that the assays do not require prior understanding of the molecular mechanism of action (MMOA), and activity in such assays might be translated into therapeutic impact in a given disease state more effectively than in target-based assays, which are often more artificial. A disadvantage of phenotypic screening approaches is the challenge of optimizing the molecular properties of candidate drugs without the design parameters provided by prior knowledge of the MMOA.

You will note that this is related to some comments I made previously about mouse models of depression.

The authors found that 28 of the first-in-class new molecular entities (NMEs) were discovered via phenotypic screening, 17 via target based approaches and 5 via making synthetic mimics of existing natural compounds. To give you a flavor of what phenotypic screening means:

For example, the oxazolidinone antibiotics (such as linezolid) were initially discovered as inhibitors of Gram-positive bacteria but were subsequently shown to be protein synthesis inhibitors that target an early step in the binding of N-formylmethionyl-tRNA to the ribosome

and for target based approaches:

A computer-assisted drug design strategy that was based on the crystal structure of the influenza viral neuraminidase led to the identification of zanamivir

The authors even ventured to distinguish discovery approaches by disease area:

Evaluation of the discovery strategy by disease area showed that a phenotypic approach was the most successful for central nervous system disorders and infectious diseases, whereas target-based approaches were most successful in cancer, infectious diseases and metabolic diseases

Unsurprising of course, given that our state of understanding of nervous system disorders is, to most viewers, considerably less complete in comparison with some other health conditions. You would expect that if there are multiple targets or targets are essentially unknown, all you are left with are the predictive phenotypic models.

Of the follower drugs 51% were identified by target based discovery and 18% by phenotypic screening. This is perhaps slightly surprising given that in the cases of the me-too drugs, you would think target-based would be more heavily dominant. Perhaps we can think of a drug which initially looked to have property X that dominated but then in the phenotypic screening, it looked more like a property Y type of drug.

The authors take on this is that it is slightly surprising how poorly target-based discovery performed within a context of what they describe as a period in which there was a lot of effort and faith placed behind the target-based approaches. I suspect this is going to be in the eye of the beholder but I certainly agree. I can’t really go into the details but there are areas where my professional career is…affected, let us say…by the smart/dumb axis of drug discovery. It should be obvious to my longer term readers that I align most closely with animal models of various things related to health and neurobiology and so therefore you may safely conclude that I have a bias for phenotypic screening. And even in the case of the target-based discovery:

at least three hypotheses that must be correct to result in a new drug. The first hypothesis, which also applies to other discovery approaches, is that activity in the preclinical screens that are used to select a drug candidate will translate effectively into clinically meaningful activity in patients. The other two hypotheses are that the target that is selected is important in human disease and that the MMOA of drug candidates at the target in question is one that is capable of achieving the desired biological response.

Right. You still need good phenotypic models and ultimately you are going to have to pass human clinical trials. The authors further worry that this higher burden, especially knowing the MMoA is going to lead to some misses.

in the case of phenotypic-based screening approaches, assuming that a screening assay that translates effectively to human disease is available or can be identified, a potential key advantage of this approach over target-based approaches is that there is no preconceived idea of the MMOA and target hypothesis.

Ultimately I think this review argues quite effectively for an “all hands on deck” approach to drug discovery but it can’t help but come off as a strong caution to the folks that think that “smarter” (aka, “rational drug design”) is the only solution. Yes, this points the finger at Francis Collins’ big thrust for a new translational IC at the NIH but also at the BigPharma companies that seem to be shedding their traditional models-based, phenotypic discover research units as fast as they can. No matter which side you come down on, this is a great read with lots to think about for those of us who are interested in the discovery of new medicines.
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Swinney, D., & Anthony, J. (2011). How were new medicines discovered? Nature Reviews Drug Discovery, 10 (7), 507-519 DOI: 10.1038/nrd3480

David Nichols on the impact of his scientific work with recreational drug users

Professor David E. Nichols is a legend for scientists who are interested in the behavioral pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, aka, ‘Ecstasy’). If you look carefully at many of the earlier papers (and some not-so-early) you will see that people obtained their research supply of this drug from him. As well as much of their background knowledge from publications he has co-authored. He has also worked on a number of other compounds which manipulate dopaminergic and/or serotonergic neurotransmission, some of which are of great interest to those in the recreational user community who seek (ever and anon) new highs, particularly ones that might be similar to their favorite illicit drugs but that may not currently be controlled. Those who are interested in making money supplying the recreational consumer population are particularly interested in the latter, of course.

Professor Nichols has published a recent viewpoint in Nature in which he muses on the uses to which some of his work has been put:

A few weeks ago, a colleague sent me a link to an article in the Wall Street Journal. It described a “laboratory-adept European entrepreneur” and his chief chemist, who were mining the scientific literature to find ideas for new designer drugs — dubbed legal highs. I was particularly disturbed to see my name in the article, and that I had “been especially valuable” to their cause. I subsequently received e-mails saying I should stop my research, and that I was an embarrassment to my university.

I have never considered my research to be dangerous, and in fact hoped one day to develop medicines to help people.

As with most scientists, I have little doubt. And ultimately, I agree with his observation that

There really is no way to change the way we publish things, although in one case we did decide not to study or publish on a molecule we knew to be very toxic. I guess you could call that self-censure. Although some of my results have been, shall we say, abused, one cannot know where research ultimately will lead. I strive to find positive things, and when my research is used for negative ends it upsets me.

It is unfortunate that Professor Nichols has been put in this position. Undoubtedly John Huffman of JWH-018 fame (one of the more popular synthetic full-agonist cannabinoids sprayed on herbal incense products) feels much the same about his own work. But I suppose this is the risk that is run with many lines of basic and pre-clinical work. Not just recreational drug use but even therapeutic use- after all off-label prescribing has to start somewhere. And individual health (or do I mean “health”) practices such as high-dosing on blueberries or cranberries, various so-called “nutritional supplements”, avoiding certain foods, exercise regimes, diets, etc may be based on no more than a single scientific paper, right?

So we should all feel some bit of Professor Nichols’ pain, even if our own work hasn’t been mis-used or over-interpreted…yet.

UPDATE: Thoughts from David Kroll over at the cenblog home of Terra Sigillata.

Women’s Health irritates the scientist-reader

Scicurious picked up an issue of Women’s Health magazine while working out at the gym. She grew gradually unamused.
Go Read. and Read. and Read.

Repost: The Women of MDMA Research

There is a #womeninscience meme bouncing around the Twitts today. Click the link and you’ll see some of the conversation, even if you are not a habitual Twitter user. Please consider joining in with an observation about, well, anything related to the life of women in science. On the Twitts, on your blog, Facebook or in the comments here or elsewhere.

I have an older post that I wrote some time ago to introduce some of the women who have contributed to the science that I talk about on the blog. This post originally appeared 24 Jul 2008.

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A comment left by a reader some time ago took exception to one of my posts highlighting another blogger.

wow, that is some excellent PR for a grad student to get for free. perhaps you could spotlight a female grad student as well…?

The ensuing discussion planted the idea for this post.

MDMA for PTSD: The first peer-reviewed clinical trial report

My readers will recall that I have blogged now and again about ongoing efforts to get 3,4-methylenedioxymethamphetamine (MDMA), the psychoactive compound preferentially sought as Ecstasy in recreational users, approved as a medication to be used in psychotherapy. The initial attempts have focused on the treatment of Post-Traumatic Stress Disorder. PTSD is a seriously debilitating condition and we may not have sufficient resources and knowledge to deal with, e.g., an anticipated uptick due to the current wars that the US is prosecuting.
I introduced the MDMA/PTSD Phase I clinical trials here, noting

The short version of the theory is that the subjective properties of MDMA (empathic, inhibition lowering, etc) are consistent with helping people in difficult psychotherapeutic situations (such as for post-traumatic stress disorder (PTSD) and, supposedly, end stage cancer anxiety) make therapeutic breakthroughs during a limited number of treatment sessions of talk therapy. This is not proposed as a chronic medication like a selective serotonin reuptake inhibitor (SSRI). The funny thing is, I approve of the concept of moving forward with clinical trials based on the available evidence.
Why not? I mean PTSD can be a very devastating psychological issue and if there are treatment-resistant cases that can benefit from a limited number of MDMA exposures, great.

I concluded that particular post with this observation.

As is general practice in medicine, sometimes there are going to be risks associated with therapy. Sometimes quite substantial risks can be acceptable if the alternative is bad. However we get ourselves into a world of trouble, sometimes even losing a perfectly helpful medication, if we are not as honest as possible, up front, over the actual risks.