# The curious question of Epibatidine



## elmoisfive

Over the past two years since I started keeping dart frogs, I've seen reference to the case of the discovery of epibitadine and linking that discovery to both new pain agents and also charges of biopiracy. Over time I've done some research on the side looking into the situation, mostly out of curiosity and have decided to share what I've found and concluded from the situation with the board. I apologize in advance for the deeper plunge than normal into technical aspects but I believe that some of the misunderstanding in this case derives from an inadequate grasp of the nuances in this situation.


*Part One - Nicotinic Acetylcholine Receptors*

First I'll give a background on the nature of biochemical system involved, the nicotinic acetylcholine receptor group. Acetylcholine receptors are a family large protein structures that span cell membranes. As a group they function to control ion flow across membranes and are activated by small molecules, called ligands that bind to them. Acetylcholine receptors can be divided into two main families, the nicotinic and muscarinic groups, named so because of the ability of nicotine and muscarine, a molecule derived from certain mushrooms to mimic the action of the endogenous ligand, acetylcholine, on these receptors. Our discussion is focused on the nicotinic acetylcholine receptor or nAChR group.

The structure of a typical nAChR is shown below. As you can see, there is an arrangement of 5 receptor subunits (protein chains) in a pentamer formation. The structure in the pink box is a representation of each of the 5 receptor subunits. Basically think of a 5 sided barrel sticking through the cell membrane with a specific orientation. 











Binding of the naturally occurring ligand, acetylcholine or the plant alkaloid, nicotine, shown below, causes the central channel to open and allows ions such as sodium and in some cases calcium to transit an otherwise impenetrable membrane. This in turn causes a depolarization (excitation) of the cell due to the change in the membrane potential (electrical charge difference across the cell membrane) and activates a variety of responses. These responses include the activation of other ion channels and also the release of other neurotransmitters. 









Acetylcholine









Nicotine

Activation of nAChRs produce a variety of physiological responses including effects on cognitive performance, locomotor activity, respiration and cardiovascular function, brain activity and brain blood flow and pain perception. The question of how such diverse responses can be modulated by this receptor system will be covered in Part Two. However even in the absence of a full understanding of this area of biology, intense research was underway in universities, government labs and the private sector. This interest was fueled by multiple purposes, one being the desire to understand the polypharmacy (multiple biological effects) of nicotine in mammals, another to see if nicotine or related compounds could be used therapeutically and thirdly because of the interest in nicotine as an insecticide. Thousands of chemicals related to nicotine (nicotine analogs) were synthesized and tested, all in the absence of an understanding of the nAChR family and also prior to the elucidation of epibatidine's molecular structure.


Glossery:

analog - closely related molecule to compound of interest
ligand - molecule capable of binding to a protein target and eliciting a biological response
nAChR - nicotinic acetycholine receptor


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## elmoisfive

*Part Two - The Molecular Diversity of the nAChR family revealed*
In the early 1980s, a series of studies and subsequent publications from a Japanese research team working with the electric ray, Torpedo californica, elucidated the key features of the molecular architecture of the acetylcholine receptor (Ref 1-3). These seminal papers paved the way for the next decade of research into the structure and function of other vertebrate acetylcholine receptors, including those of humans. 

So we return to the question from Part One. How can such a wide variety of physiological responses be controlled by a 'single' receptor system? The answer lies in the diversity of nAChRs and their distribution in the body. nAChRs consist of alpha, beta, delta and gamma/epsilon chains (subunits). There are nine forms of alpha chain, alpha-2 through alpha-9 and three beta chains, beta-2 through beta-4. The neuronal forms of nAChRs consists of either combinations of alpha and beta chains (subunits) or pure alpha chains in the case of alpha 7, 8 and 9. In contrast, the muscle form of nAChR has is composed of two alpha, a beta, a delta and either a gamma or epsilon chains. The various receptor subunit combinations respond with differing sensitivities to agonists such as nicotine. In addtion, different forms of nAChR are expressed in various tissues in the body.

A brief listing of some the major receptor subtypes, their location(s) and associated activities is shown below

--------------------------------------------------------------------
*Brain*

Receptor subtype = Alpha4,beta2 
Tissue distribution = Cerebral cortex, Thalamus, Hippocampus	
Effects = Cognition, Memory, Analgesia

Receptor subtype = Alpha7 
Tissue distribution = Cerebral cortex, Hippocampus
Effects = Learning/memory

Receptor subtype = Alpha3,beta4 
Tissue distribution = Interpenducular nucleus, Habenula
Effects = Reward (addiction)

Receptor subtype = Alpha6,alpha3,beta2	
Tissue distribution = Nucleus accumbens 
Effect = Reward (addiction)

Receptor subtype = Alpha4, beta4 
Tissue distribution = Hippocampus, Chemoreceptor Trigger Zone	
Effects = Seizures, Emesis

*Autonomic ganglia* 

Receptor subtype = Alpha3, beta2 and Alpha3, beta4	and Alpha3, beta4, alpha5
Tissue distribution = Heart, Gastrointestinal tissue 
Effects = Vasoconstriction, GI distress

*Skeletal muscle* 

Receptor subtype = Alpha1, beta1, delta, epsilon	
Effect = Respiratory paralysis
------------------------------------------------------------------

In addition to the elegant dissection of the physiology of the various receptor subtypes, recombinant cell lines expressing these various receptor combinations had been constructed and were being used to screen chemical compounds for activities by the early 1990s led by groups such as SIBIA Neurosciences (spin out from the Salk Institute) (Ref 4,5) 

Publications and patents were also starting to issue showing attempts to capitalize on the new knowledge surrounding the diversity and distribution of nAChRs. The first significant Abbott patent, US Patent 5,278,176 (Ref 6) was filed in 1992. It is important to note that 2-4 years of research typically precedes the filing of this kind of patent due to the preparation and testing of a large number of molecules. So this suggests significant activity by Abbott in the years proceeding 1992 in this area. Interestingly, this patent also cites as prior art a 1982 Philip Morris patent on the preparation of nicotine analogs demonstrating the long history of work in this arena. 

Abbott Laboratories first venture into the clinic with a molecule was with ABT-418. This compound showed some favorable selectivity for alpha4,beta2 receptor subtype versus alpha7. In preclinical studies it demonstrated improvements in cognitive function and it was subsequently tested for utility in humans across Alzheimer's, Parkinsons disease and attention deficit hyperactivity disorder (ADHD). While it showed promise in the clinic, it's short half life required that the drug be administered via a dermal patch.










*References*

(1) Primary structure of α-subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence. Nature 299, 793 - 797 (28 Oct 1982) 

(2) Primary structures of β- and δ-subunit precursors of Torpedo californica acetylcholine receptor deduced from cDNA sequences. Nature 301, 251 - 255 (20 Jan 1983)

(3) Structural homology of Torpedo californica acetylcholine receptor subunits. Nature 302, 528 - 532 (07 Apr 1983)

(4) US Patent 5,369,028 "DNA and mRNA encoding human neuronal nicotinic acetylcholine receptor compositions and cells transformed with same" Filed April 3, 1990. Inventors Michael Harpold and Steven Ellis. Assignee: The Salk Institute Biotechnology/Industrial Associates, Inc

(5) US Patent 5,371,188 "Neuronal nicotinic acetylcholine receptor compositions" Filed June 12, 1992. Inventors Stephen Heinemann et al. Assignee: The Salk Institute for Biological Studies 

(6) US Patent 5,278,176 "Nicotine derivatives that enhance cognitive function" Filed August 21, 1992. Inventor Nan-Horng Lin. Assignee: Abbott Laboratories


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## elmoisfive

*Part Three - Epibatidine Discovered and the Controversy Begins*

In 1974, John Daly and coworkers at the National Institutes of Health collected frogs from the Pacific highlands of Ecuador. Back in their labs, they extract alkaloid containing material from the skin of E. tricolor and upon injection into mice, note that the mice arch their tails, a characteristic that had previously been associated with opioid treatment. Unfortunately the small amounts of the extract produced were insufficient to determine the structure. Captive bred frogs did not produce the substance of interest leading to speculation that dietary influences in the wild were important. The subsequent protection of E. tricolor under CITES *[correction - CITES was not the reason for the inability to acquire further material from wild caught frogs - see Blort's post below]* prevented further acquisition of specimens so elucidation of the structure of this unknown compound awaited refinements in analytical technology. 

With improvements in analytical techniques, members of Daly's lab were able to determine the structure of this substance which they named epibatidine (shown below) and published their results in 1992 in the Journal of the American Chemical Society (Ref 1)

An examination of the structures of epibatidine and nicotine show a striking resemblance...in fact that they are closely related in a chemical sense.










I have circled two key chemical motifs in the next picture....the pyridine ring in red and the amine (N atom) functional group in blue. These features are found in many known nicotine compounds.










In this next picture I have circled the key pharmacaphore or active core in green. Note that nicotine and epibatidine are almost identical, the lack of identity due to the isosteric difference in the pyrrole ring (the 5 membered ring on the left containing the nitrogen N).










So while epibatidine is certainly an interesting molecule and has some interesting properties (discussed below), it is very closely related to a known compound, nicotine. I believe the lack of understanding of the close relationship of epibatidine to known compounds such as nicotine is perhaps one cause for the belief that it somehow enabled an entire field of drug discovery.

The close chemical relationship to nicotine was immediately noted by Daly and others and subsequent investigation revealed that epibatidine worked via interaction with nicotinic acetylcholine receptors (Ref 2,3). While the Daly paper (Ref 2) provides confirmation that epibatidine is in fact a nicotinic agonist, the Abbott publication (Ref 3) presents a detailed pharmacological characterization both in vitro and in vivo of epibatidine. Interestingly, they demonstrated that epibatidine had none of the anxiolytic activity demonstrated by compounds such as ABT-418 though at the time it was unclear whether this was due to differing mechanisms of action or toxic side effects of the compound. They also noted dose limiting toxicities such as hypothermia and motor incoordination that prevented dosing at higher levels.

However, being intrigued by the positive pharmacology seen with epibatidine and because they already had a significant research focus on nicotinic agents, they sought to tease away the negative activity due to epibatidine's interaction with non central nervous system nAChRs in sympathetic ganglia and the neuromuscular junction. The end result of this first wave of effort was the synthesis of ABT-594.










While ABT-594 demonstrated similar potency at the brain alpha4,beta2 receptor subtype, it was 4000 times less active at the neuromuscular nicotinic receptor than epibatidine, thus presenting an improved therapeutic index (desired activity versus undesirable activity). Without the dose limiting toxicities of epibatidine, researchers were able to demonstrate activity of ABT-594 in both acute and neuropathic pain. 

In human clinical tests, initial results were encouraging for ABT-594. However it's development was subsequently halted as more extensive studies demonstrated unacceptable side effects, primarily gastrointestinal related. Subsequently, it appears that interest in working with this particular chemical scaffold has waned. presumably due to the inability to widen the therapeutic index by dialing out the gastrointestinal effects..

Abbott has partnered with the Danish biotech company, Neurosearch, in their effort to define next generation agents. One of these is ABT-894 -the definitive structure of this molecule is not publically known. However, a review of Neurosearch publications (Ref 4,5) and related patent filings reveal the familiar nicotine motif of pyridine ring + (linker) + a nitrogen containing ring system.

In addition, significant work by other organizations has continued in the nicotinic agent field, fueled both by the potential utility of agents and the biological tools now available to researchers. Other molecules of potential interest include SIB 1508Y (alpha4,beta2 selective), GTS-21 and TC-1734. GTS-21 was patterned off the invertebrate toxin, anabasine and demonstrates overlapping activity at both alpha 7 and alpha4,beta2 receptors yet functionally acts as a partial alpha7 agonist. TC-1734 from Targacept has good affinity for alpha4,beta2 receptors (Ki = 11 nanomolar) but poor to no interaction with alpha7, ganglionic or neuromuscular nicotinic receptor subtypes.



























References

(1) Spande, T. F., H. M. Garraffo, M. W. Edwards, H. J. C. Yeh, L. Pannell, and J. w. Daly. Epibatidine: a novel (chloropyridyl)azabicycloheptane with potent
analgesic activity from an Ecuadoran poison frog. 
J. Am. Chem. Soc.114:3475-3478 (1992).


(2) Epibatidine, a Potent Analgetic and Nicotinic Agonist

BARBARA BADIO and JOHN W. DALY
Mol. Pharmacol. 45:563-569 (1994)

(3) (±)-Epibatidine Elicits a Diversity of In Vitro and In Vivo Effects Mediated by Nicotinic Acetylcholine Receptors
JAMES P. SULLIVAN, MICHAEL W. DECKER, JORGE D. BRIONI, DIANA DONNELLY-ROBERTS, DAVID J. ANDERSON, ANTHONY W. BANNON, CHAE-HEE KANG, PAMALA ADAMS, MARIETTA PIATTONI-KAPLAN, MICHAEL J. BUCKLEY, MURALI GOPALAKRISHNAN, MICHAEL WILLIAMS and STEPHEN P. ARNERIC
The Journal of Pharmacology and Experimental Therapeutics 271: 624-631 (1994)

(4) Novel Potent Ligands for the Central Nicotinic Acetylcholine Receptor:
Synthesis, Receptor Binding, and 3D-QSAR Analysis. J. Med. Chem. 43: 2217-2226 (2000)

(5) New Ligands with Affinity for the alpha4,beta2 Subtype of Nicotinic Acetylcholine Receptors. Synthesis, Receptor Binding, and 3D-QSAR Modeling. J. Med. Chem. 49: 3159-3171 (2006)


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## elmoisfive

*Part Four: What role has epibatidine played in nicotinc agent research?*

The elucidation of the structure of epibatidine was preceded by significant progress in the area of nicotinc agents so to claim that it played the seminal role some assign to it in this arena is somewhat overreaching. However it is fair to say that it's unique pharamcological profile and arrival at the time that receptor subtype profiling was becoming readily available assisted in our understanding of the area. Unfortunately research into improving upon its activity proved unsuccessful. A useful research tool yes but also a fairly active rat poison. Other lines of research that were active at the time of its discovery (structural elucidation wise) have dominated the field and epibatidine is slowly being relegated to sidelines.

Ironically the first approved nicotinic agent, varenicline, from Pfizer that was approved earlier this year for smoking cessation was derived from the naturally occurring compound, cytisine. Cytisine is an alkaloid found in plants such as Common Broom and Mescalbean. 

Varenicline is a partial agonist at the alpha4,beta2 receptor and appears to reduce nicotine craving without being rewarding (addictive).









Varenicline









Cytisine

Thus concludes my analysis of the epibatidine situation. I would note that the opinions expressed are mine alone and I'm offering one particular perspective. Others may chose to disagree with my conclusions but I thought it would be helpful to provide a fuller perspective on the story than one usually reads in summary articles.

Comments, questions, challenges are welcome as always.

Bill


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## Homer

Thanks, Bill. Great background info, history, and clarification by comparing the chemical structures.

I have read some of the links to allegations of biopiracy purportedly drafted by governmental agencies from South American countries and read some articles related to the epibatidine discovery and debate.

I must admit, most of the allegations and official statements I have read just make me chuckle.


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## Blort

Hi Bill,

Wow! Wonderful post. Unfortunately my Chemistry knowledge stops at Chem 101. I have tangential questions though:



> "The subsequent protection of E. tricolor under CITES prevented further acquisition of specimens so elucidation of the structure of this unknown compound awaited refinements in analytical technology."


What is the source for this? In 1987 there was a shipment to the US according to CITES with no usage code, in 1989 a shipment to Germany with a science code, from 1994 to 1997 a couple thousand for trade, and in 2000 a handful for trade. The reason I ask is that I often hear this claim leveled against CITES by scientists. I find your phrasing confusing as to what the limitation was. Was it the acquisition of specimens, refinements in analytical technology, or both?

The other treaty beyond CITES whose role I don't yet understand is the Convention in Biological Diversity. I think it, not CITES, touches on issues of technology transfer, bio-rights, etc.

Next, is the Brazilian article's claim that castis may also have pharmaceutical value have any basis in fact regarding epibatidine?

Finally, I will see if Justin Yeager can post on here or to you in private about some of what he learned while in Ecuador. He worked with scientists in the lab, not just government enforcement agencies, and epibatidine was a subject of some concern I believe.

Homer,

Those government's claims may be full of rhetoric and not ground in much science, but I think they raise difficult, abstract issues such as national sovereignty rights, ownership of living beings, ownership of the offspring of living beings, etc. I don't think their concerns should be dismissed off hand. While their position may not be completely defensible, I have seen a fair amount of hostility bordering on arrogance from the scientific community about some sort of inalienable right for them to take whatever biological materials they want so they can do whatever research they see fit.

I think this arrogance is multiplied when in many cases it is perceived that the some in the scientific community are just swooping in, collecting animals and plants, bringing them back out of Latin America and leaving little tangible benefit to the host country beyond a few dollars spent on incidentals and paying a guide while in country. I think some of this arrogance combined with overt disdain expressed about the alleged inherit corruption of Latin America which paints all government employees as bungling, self-serving bureaucrats can easily appear racist or imperialist. I'm not claiming that corruption isn't an issue, but I have read enough reports from these country's government authorities to know that there are a lot of caring concerned people out there. I also find another suggestion that was alluded to in the mysto thread problematic. The claim seemed to be that because these governments hadn't done enough in the past to preserve habitat, they had somehow forfeited their current rights to exert their political and legal ability to exert pressure on what they see as violations of their national laws and international treaties.

The history of Latin American governments is checkered, and I don't want to derail Bill's thread, but western involvement in Latin America, Manifest Destiny, policies like the Washington Consensus, the backing of corrupt, fascist governments by American power, and a host of other issues are part of the complete geopolitical picture (wasn't a chem major, but was a Latin American studies one).

Marcos


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## elmoisfive

Marcos,

I confess that the CITES database has not been very user friendly to me so my statement that CITES shut the door on further acquisition of E. tricolor by Daly's group is probably incorrect. This was one part of the data trail that I even hesitated to dive into because of the potential to harm reputations, etc. So I'll make a correction in that part of the post. I certainly am not one to slam CITES as it does tremendous good when used properly.

What is factual is that the analytical technology of the day (mid 70s) was not capable of rendering accurate structure determination of the small amount of material that was isolated by Daly's group. However by the mid to late 80s, the technology had advanced, particularly nuclear magnetic resonance technology, to the point where working with fairly small sample sizes was less of a problem.

I would love to have Justin weigh in with his perspective on the situation. I miss his posts on the board.

The question of biopiracy is multifaceted and quite complex. I agree that there have been cases where materials were collected under less than desirable circumstances. In this particular case, I think that the part that is most difficult to dissect is under what circumstances Daly acquired the E. tricolor. The more interesting question is one of if Daly had elucidated the structure of epibatidine and it was this hideous intractable compound that no one in their right mind would consider trying to use as a drug, would there have been a fuss? Usually controversy on biopiracy goes hand in hand with the belief that money is about to be made by someone.

It seems that these issues occur when animals or plants are involved. What most people don't know is that many of the antibiotics that they took as children or even today had their origins in soil samples collected from around the world and their microfauna cultured, particularly _Actinomycetes_, to see what interesting secondary metabolites were produced. Missionaries, business travelers, students, native peoples were all called upon to collect a bit of dirt from various locations, put it in an envelope and mail it off to interested parties. In some cases, cultures were even obtained by extracting materials off letters sent from around the globe. A surprising number of very powerful antibiotics were discovered and developed using this fairly simple approach.

So here we have the case of where 'dirt' from various countries produced drugs. I am surprised that some wag hasn't proposed an international convention on the transport of dirt to protect counties' rights to the national soil heritage :? 

I'll have to look into the question of castis vis-a-vis their pharmacopeia and respond later.

Lastly, I'm the fellow who suggested somewhat tongue in cheek in the mysti thread that Brazil by their blatent ecoterrorism has lost some of their moral standing with me....kind of like the United States preaching to other countries on the matter of greenhouse gases while we burn fossil fuels at an incredible rate :roll: Brazil has a path to resolution vis-a-vis the contract issue with the museum involved. But this constant 'we was robbed - oh woe is us' sob story while simultaneously torching the Amazon just leaves a bad taste in my mouth.

Now if we were talking about a country like Costa Rica that has a pretty decent track record on ecological issues, I'd give them a lot of my time even if I didn't agree with their position. But the Brazilians given their over four decade old policy of 'opening the Amazon up for development', a policy that has been sustained through military dictatorship and democracy, right and left wing governments....ummm I don't think so. I tend to get a bit worked up on the issue to the point that I'm now derailing my own thread 

But anyway, back to the question of epibatidine. I remember when I first saw the structure wondering what all the ruckus was about....so it's a nicotine homolog, albeit a fairly interesting one since it was found in an animal. After all the structure of nicotine has been known for over 150 years now and the first chemical synthesis occurred over 100 years ago in the late 1890s. Even the chloro-pyridine motif, while being unusual in a biologically derived material, is pretty common fare in medicinal chemistry since a chlorine is isosteric with a methyl group in that context and is used to alter the electronic properties of the pyridine ring, impact metabolic handling of the drug, etc. So I'm like hmmmm well if the structure of epibatidine was uncovered shortly after it was isolated, i.e. mid 70s, it would have been a seminal discovery. But coming when it did landed it smack dab in the middle of the nicotinic receptor revolution and it was swept up in the current. 

Bill


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## Blort

Bill,

I hear you on bio-piracy issues and such. Those are topics worthy of their own thread some day.

Part of what I understood the hype was about revolved around addiction to epibatidine with the drug being touted as non non-addictive alternative to morphine. Was that totally overblown and do you anticipate that much of the buzz around epibatidine is likely to fade?

Marcos


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## elmoisfive

Marcos,

Yes the speculation is that one can produce non-addictive nicotinic agents that would function for pain, cognition, etc. Preclinically, results with ABT-594 were suggestive that it did not produce dependency in rat studies. However, these types of experiments are difficult to extrapolate to the human setting and ABT-594 did not progress far enough in clinical testing to give the hypothesis a good test.

Varenicline as mentioned before has been shown to have efficacy in the absence of dependency (addiction). However, there is some speculation this may be due to the fact that it is a partial as opposed to full agonist at the nicotinic acetylcholine receptor. A partial agonist activates a receptor but only produces a partial physiological response compared to a full agonist.

Epibatidine and ABT-594 are full agonists and it is extremely difficult although not impossible to 'dial' partial agonism into a structural class. So if the partial agonist theory pans out, it will be one more strike against those compounds. On the other hand, there is too little data to draw a conclusion at this point.

I think that the buzz surrounding the opportunities of selective nicotinic agents will continue to be with us and in time the field will produce more drugs. How Epibatidine continues to be tied to this situation will likely be more due to popular imagination as opposed to therapeutic utility.

Bill

P.S. I found some info on the other questions you posed and will post that later this evening.


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## Homer

Blort said:


> Homer,
> 
> Those government's claims may be full of rhetoric and not ground in much science, but I think they raise difficult, abstract issues such as national sovereignty rights, ownership of living beings, ownership of the offspring of living beings, etc. I don't think their concerns should be dismissed off hand. While their position may not be completely defensible, I have seen a fair amount of hostility bordering on arrogance from the scientific community about some sort of inalienable right for them to take whatever biological materials they want so they can do whatever research they see fit.
> 
> . . . .
> 
> Marcos


Marcos, I would not be surprised that certain aspects of the scientific community may have been arrogant in their approach to discovery, but I really cannot comment on the general sentiment in the scientific community. However, what I see as comical is not only the lack of grounding in science, but a lack of planning on a legal/political level to protect such discoveries, and a lack of grounding in any coherent legal basis when the government does speak.

Exactly how do you limit the geographic boundary of ideas based upon research that may have involved a chemical from an organism that originated from a particular geographic location? Essentially, that is what the allegations of biopiracy in this matter are trying to accomplish.

"Come to Brazil, research our wildlife, but your ideas and discoveries even tangentially related thereto belong to us."

Sounds like a perfect commercial for expanding investment in the Brazilian ecosystem. 

I agree that the issue is extremely complex (if not impossible) if your aim is to ensure that the developing countries receive a vested interest in any intellectual property that is derived from their environment while encouraging exploration and exploitation of that intellectual property. I just don't believe that this is a goal that truly exists amongst the international community.


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## elmoisfive

I dug up several additional sources of information, two of them being scientific publications by Daly and one of them being a rather inflammatory commentary from Ecuador (credit to Alan Zimmerman for digging the latter up and posting it a short while back).



> The presence of epibatidine might not have been suspected if an aliquot of the methanolic extract from an Ecuadorean frog collected in 1974 had not been injected into a mouse in a routine toxicity assay. The mouse was observed to arch its tail over its back, a drug reaction known as the Straub tail. Excited and suspecting an opioid alkaloid, a return with Myers to the collection site yielded several hundred frogs. The Straub tail provided an initial assay for isolation of an alkaloid that, after structure elucidation, we named epibatidine. It was a trace alkaloid in the extracts at levels of 1 microgram per frog. Structure elucidation by NMR spectral analysis of an N-acetyl derivative was finally accomplished in 1992. (Ref 1)





> U.S. National Institutes of Health (NIH) scientist John Daley there are identified the chemical structure of the frog's secretion, thanks to the information given to him by indigenous and local communities about the physiological effects of this substance. To isolate the activates principle, there am illegally obtained to sample of 750 frogs which we believe left Ecuador by diplomatic pouch since there is not proof that the Ecuadorian Institute of Natural Forests and Wildlife (INEFAN) granted to for license the frog to be commercially expoited. License This kind of is to BASIC requirement of Ecuadorian law, since the frog appears in the annex to the Convention on International Trade in Endangered Species (CHALLENGES), to which Ecuador is party since 1975.(Ref 2)


Well they both agree that he took hundreds of frogs out of the country.



> Furthermore, INEFAN banned any bioprospecting activities involving this species in 1996. Yet it was through such bioprospecting that Abbot Laboratories in the United able States was to market its drug ABT-594, derived from epibatidine. ABT-594 is an analgesic two hundred times dwells potent than morphine. Abbott Laboratories obtained the patent on epibatidine to after Daley isolated it. (Ref 2)


You know this paragraph is ridiculous on several levels. How Abbott Laboratories gets connected to this 'supposed act of biopiracy' 20 years after the fact is a bit of a mystery to me other than the fact that they read the scientific literature and decided to investigate the behavior of epibatidine. Something scientists in academia and industry do on a daily basis. In addition, it was Daly and co-workers who patented epibatidine and related compounds for use in pain not Abbott Labs. (Ref 3,4). Who is the owner of those patents? Why the United States government. 

Furthermore, ABT-594 came no where near being marketed. All I can say about Ms. Bravo and Ms. Gallardo is that they aren't particularly tuned into the facts. So I'm not sure I put much stock in the frog smuggling part of their story.

To your question Marcos about epibatidine being present in other dart frogs....according to Daly epibatidine has a fairly restricted distribution even within E. tricolor. So it is unlikely to be found within other dart frogs such as castaneoticus.



> epibatidine, which was detected in “significant” amounts only in two populations of Epipedobates tricolor but was absent in several other populations in Ecuador(Ref 1)


The dietary source of epibatidine discussed....



> For epibatidine, sites in Ecuador, where epibatidine was found in the frog Epipedobates tricolor, would most likely provide the dietary source. At one site, epibatidine was present at about 1 microgram per skin, while at a nearby riparian site it was present at only 0.1 micrograms per skin. It appears likely that a food chain from a plant may pertain. Thus, a plant may produce the nicotine-like epibatidine, which is taken up by an arthropod and then passes to the frog. (Ref 1)


Now switching to a later review article that Daly and coworkers wrote (Ref 5 - a really good source of info on amphibian toxins btw), some other interesting info....



> Three other pyridine alkaloids have been detected in skin extracts of dendrobatid frogs. These are nicotine, noranabasamine, and a pyridylnicotine. Nicotine has been detected in both dendrobatid and mantellid frogs, but very rarely and only in trace amounts. Noranabasamine was isolated from a Colombian
> poison-dart frog, while the pyridylnicotine was detected as a trace alkaloid in extracts of another Colombian
> dendrobatid frog, Dendrobates lehmanni (unpublished results)(Ref 5).





















Okay that is very interesting and serves to tie epibatidine even more closely into the whole nicotine story. But here is the part I predict will cause even more "political trouble" 



> In addition, a structurally related alkaloid, phantasmidine. recently isolated from skin extracts of a population of an Ecuadorandendrobatid frog (Epipedobates tricolor), is under investigation (Fitch et al., unpublished results). The frog has been called the “phantasmal poison-arrow frog”. The
> empirical formula for phantasmidine is C11H11N2OCl. A partial structure has been defined by NMR spectroscopic analysis (unpublished data)...It is a potent nicotinic agonist. (Ref 5)












Oh bleep me running. Forget Epibatidine, we've found the true miracle drug from E. tricolor. Unfortunately the structure is incomplete. Oh well I have a good idea of the structure and I suspect Dr. Daly does as well... 

Perhaps the biggest tragedy of this whole situation is how John Daly has been dragged through the mud on this one. I don't know the man but I know his type, he is a true blue scientist who lives for data and has a passion for what he does. He comments in reference one on the difficulties of gaining permission to do field research and laments the fact that precious ecological niches are being destroyed along with their associated biodiversity. Yep a real nasty 'pirate'...worried about little frogs and bugs and plants. Since I share his concerns I suppose I'm a pirate as well. 

Bill 


References

(1) "Ernest Guenther Award in Chemistry of Natural Products. Amphibian Skin: A
Remarkable Source of Biologically Active Arthropod Alkaloids" John Daly
J. Med. Chem. 46: 445-452 (2003)

(2) AUTHOR: Elizabeth Bravo & Lucia Gallardo, Acción Ecológica PUBLICACION: contribución a BIO-IPR 
PUBLICATION: submitted by the authors to BIO-IPR FECHA/ 
DATE: Noviembre/November 1998 FUENTE/ 
SOURCE: Acción Ecológica, Ecuador 

(3) US Patent 5,314,899
Epibatidine and derivatives, compositions and methods of treating pain 
Inventors: John W. Daly, Thomas F. Spande and Hugo M. Garraffo
Assignee: The United States of America

(4) US Patent 5,462,956 (this is a division of the 5,314,899 patent)
Epibatidine and derivatives, compositions and methods of treating pain 
Inventors: John W. Daly, Thomas F. Spande and Hugo M. Garraffo
Assignee: The United States of America

(5) "Alkaloids from Amphibian Skin: A Tabulation of Over Eight-Hundred
Compounds" John W. Daly, Thomas F. Spande, and H. Martin Garraffo
J. Nat. Products 68: 1556-1575 (2005)


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## Blort

Bill,

I do need to clarify some things:

* My comments about arrogance are not directed at anyone in particular, this is just a theme that I have picked up on various threads on some of the science forums and mailing lists.

Most of the time these comments are probably in frustration or innocuous steam venting, but if I have heard them enough after 2 years to get this impression, I know I'm not the only one. I do concede that I can see a legitimate basis for this frustration. I do get concerned when this seems to be a deeply entrenched attitude. We all bring our biases to the table, and I think in amphibian conservation this can end up causing a lot of harm. For a good lesson on the polarity between science and private herpetology browse the PARC mailing list archives. Right now is an exciting time for amphibian conservation where we are close to many of these elements coming together, but I think moving forward, especially with dendrobatids, is going to require governments, private individuals, zoos, and scientists all at the table. Through various conversations I have had with members of all these entities, I can say we all have some baggage we need to work through about how we view one and other.

(tangent) I think the wholesale addition of dendrobatids to CITES was probably a mistake, but I can say that the addition of amphibians to CITES seems haphazard and sporadic at best. I think this combination (the wholesale addition of an entire genus to CITES) and the fact that since 1987 the only other addition to the appendices for amphibians were the genus mantella and Scaphiophryne gottlebeis seems to me problematic in the midst of a global amphibian crisis.

* I don't maintain that the claims of bio-piracy are valid, my point all along has been that this is an emotional issue aggravated by the possibility of some lucrative wonder drug. A big pharmaceutical company like Abbot is also easy pickings for allegations where the company serves as the proxy for some vast pharmaceutical conspiracy to exploit the Amazon.

There are many issues with CITES and it's management which are a reasonable source of frustration. That Bravo and Gallardo article is interesting, but should be taken with a grain of salt and not seen as the government's position in my opinion. From what I can see of the organization "Acción Ecológica" they are a nonprofit with a certain mission and their article should be taken in that context.

There is a fundamental flaw in their argument though, while CITES was in force in 1976 when the movement of frogs is alleged to have happened, dendrobatidae was not added until 1987. So, CITES would not apply. I haven't dug into Ecuadorian laws, but I am guessing that they probably didn't have strong export control laws on animals in the mid 70's. To say that this happened through diplomatic pouch is almost funny, it's creative theory to say the least.

I think part of the problem here is that there isn't a very unified voice about these issues yet to represent the concerns of international scientists AND of national governments and peoples of resource rich countries. I think CBD is supposed to be part of that mechanism to ensuring that there is an equitable access to global biological resources in a way that it isn't one sided. Since there doesn't appear to be a unified voice about this, those groups which are concerned (albeit maybe not unbiased) fill in the gaps.

(tangent) I think you see the same issue over pets where the most vocal groups are animal rights groups with a pretty narrow, intractable position, or industry groups. Lacking a middle ground which represents the average pet owner, that voice gets drowned out. I've seen this in testimony over shipping regulations for animals where animal rights organizations with a stated goal of eliminating pets are the ones giving testimony. So, in this case you have what appears to be silence from the key players in the issue (for whatever reason), and in the gaps you get editorials from a conservation group.

At any rate, I would note that almost all the articles about the tricolor and casti allegations that I have seen so far are really more editorials than government positions. Even in the case of the casti article I posted, that is the voice of one PM in Brazil, not necessarily the Brazilian position. As nobody should take these forum comments to represent the American perspective or that of our employers, we should be careful of doing the opposite.

Marcos


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## Homer

elmoisfive said:


> In addition, it was Daly and co-workers who patented epibatidine and related compounds for use in pain not Abbott Labs. (Ref 3,4). Who is the owner of those patents? Why the United States government.


More specifically, the Secretary of the Department of Health and Human Services, since Daly was working for the National Institutes of Health in Bethesda at the time of discovery.

Marcos, I apologize that I said Brazil when I meant Ecuador in my earlier post. My references were to the statement posted by Allan Zimmerman, which I thought to be affiliated with some government agency.

If that is not the position of the government, what postitions and statements were you referring to when you said:



Blort said:


> Those government's claims may be full of rhetoric and not ground in much science, but I think they raise difficult, abstract issues such as national sovereignty rights, ownership of living beings, ownership of the offspring of living beings, etc. I don't think their concerns should be dismissed off hand.


?


[/quote]


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## bluedart

Wow Bill, an incredibly informative read! I'll post a response worth reading later...


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## EDs Fly Meat

Hmmm I was hoping you all could get a little more detailed on the issue.

NICE POST BILL! Well done.

As someone who works in the healthcare industry I would love to see this drug created (and am not expecting to). And correct me if you already answered this question, but since it works on nicotine recepetors wouldn't patients have dangerously highly elevated heart rates and blood pressures?

I always wondered that.
Dave


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## elmoisfive

Dave,

If a compound was sufficiently devoid of activity at the ganglionic nicotinic acetylcholine receptors, in theory the risk of elevated heart rate and blood pressure would be avoided. I say in theory because the odds of obtaining a truly clean agent are remote, particularly if you are starting with a compound like epibatidine. Of course you would also want to avoid activity at the neuromuscular junction (respiratory paralysis is a bad thing!) and would also want to avoid the reward receptors that would lead to chemical dependency.

Noting that the desirable receptor targets are in the brain behind the blood brain barrier compared to the peripheral undesirable targets, if one could achieve preferential sequestration in the brain you can limit activity of the drug peripherally. This strategy (or the reverse in situations were brain activity is bad) has actually been used as the basis of developing several successful drugs. Unfortunately this is not something easy to design into a drug. 

I'm optimistic that over time the problems will be ironed out and we will see some pretty interesting drugs come to market in this arena. Molecules like TC-1734 offer possibilities...time will tell.

In addition there are efforts on pain with respect to the excitatory amino acid receptor family, various neuropeptides and even selective opioid receptor modulators. Chronic pain is a major medical issue, being the cause of considerable morbidity and in some cases mortality. 

Finally after 'slamming' epibatidine because it won't lead to a drug, I have to give it and the frog credit for its effectiveness as a poison. It is far more potent than nicotine hence the ability to do damage with a fairly small dose. One rarely sees the full spectrum of toxicities with nicotine in humans, especially respiratory paralysis although this latter activity has been seen with workers in tobacco fields in cases of significant and prolonged exposure to the plants.

Bill


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## girlfrog

> if one could achieve preferential sequestration in the brain you can limit activity of the drug peripherally.


I was going to comment that this is HARD to accomplish, but you already said that. Your right, but that hurdle is a BIG one, like Ayer's rock in Austrailia big. Great post Bill, keep ones like these coming.


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