Epibatidine

Epibatidine

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Epibatidine is an alkaloid
Alkaloid
Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms. This group also includes some related compounds with neutral and even weakly acidic properties. Also some synthetic compounds of similar structure are attributed to alkaloids...

 found on the skin of the endangered Ecuadorian frog, Epipedobates tricolor
Phantasmal poison frog
A phantasmal poison frog is a species of poison dart frog. Their natural habitat is the Andean slopes of the central Ecuadorian Bolívar province. They have radiant color, powerful poison, and yet are some of the smallest frogs around, ranging from to...

. These frogs, like other poison dart frogs, are best known for their ability to sequester poisons from their prey and secrete these poisons onto their backs. Many Amerindian tribes would swipe the frogs' backs with their blowdarts to provide a much more successful hunt. The one toxin that distinguishes the Epipedobates tricolor
Phantasmal poison frog
A phantasmal poison frog is a species of poison dart frog. Their natural habitat is the Andean slopes of the central Ecuadorian Bolívar province. They have radiant color, powerful poison, and yet are some of the smallest frogs around, ranging from to...

 from other frogs in this family
Family (biology)
In biological classification, family is* a taxonomic rank. Other well-known ranks are life, domain, kingdom, phylum, class, order, genus, and species, with family fitting between order and genus. As for the other well-known ranks, there is the option of an immediately lower rank, indicated by the...

 is epibatidine. The frog uses the compound to protect itself from predators. Animals many times larger would die from the small amounts of epibatidine that the frog secretes.
Epibatidine, which was being researched in 1974, turned out to be a very powerful analgesic
Analgesic
An analgesic is any member of the group of drugs used to relieve pain . The word analgesic derives from Greek an- and algos ....

. This had proven a powerful argument for further research, which has shown that epibatidine has gastrointestinal side effects
Side Effects
Side Effects is an anthology of 17 comical short stories written by Woody Allen between 1975 and 1980, all but one of which were previously published in, variously, The New Republic, The New York Times, The New Yorker, and The Kenyon Review. It includes Allen's 1978 O...

. This gives the compound a very small therapeutic index and it is very unlikely to ever appear on the medicinal market.

History



John Daly, working for the National Institute of Health, collected samples of skin secretions from the frog in 1974. Although it was shown to work as an effective analgesic in mice with a potency 200 times that of morphine, technology at the time was not able to discern the chemical's structure because the preserved sample was too small. In addition, Daly's research was hindered by the Convention on International Trade in Endangered Species of Wild Fauna and Flora, which put heavy restrictions on the collection of Epipedobates tricolor.

After further research in the late 90's, when technology finally allowed the determination of the compound's structure, there was great excitement when epibatidine was found to be non-opioid
Opioid
An opioid is a psychoactive chemical that works by binding to opioid receptors, which are found principally in the central and peripheral nervous system and the gastrointestinal tract...

. This was proven by Daly's experiment: administration of naloxone
Naloxone
Naloxone is an opioid antagonist drug developed by Sankyo in the 1960s. Naloxone is a drug used to counter the effects of opiate overdose, for example heroin or morphine overdose. Naloxone is specifically used to counteract life-threatening depression of the central nervous system and respiratory...

, an opioid antagonist, did not reverse epibatidine's effect. Being a non-opioid suggested that it might have been a non-addictive compound with very few adverse effects. However, because of the small difference between therapeutic and toxic dose, epibatidine has never been used in humans. Abbott Laboratories has produced derivatives of epibatidine in search of a less toxic analgesic with less side effects than opioids.

Synthesis



Several total syntheses have been devised due to the relative scarcity of epibatidine in nature. E. Tricolor frogs do not naturally possess epibatidine, but obtain epibatidine or its precursor from their surroundings. It has been suspected that the frog obtains its epibatidine or a precursor
Precursor (chemistry)
In chemistry, a precursor is a compound that participates in the chemical reaction that produces another compound. In biochemistry, the term "precursor" is used more specifically to refer to a chemical compound preceding another in a metabolic pathway....

 from is a flowery plant, since the similar toxin nicotine is also derived from this kind of source. Locating the source is important, since epibatidine recovery from E. Tricolor frogs is made illegal after the frogs had been declared endangered. The population is still very unstable, and will not allow scientists to extract epibatidine in the near future.

After the discovery of epibatidine's structure, more than fifty ways to synthesize epibatidine in the lab have been found successful. After a nine-step procedure, the substance was obtained as a racemate. The enantiomer
Enantiomer
In chemistry, an enantiomer is one of two stereoisomers that are mirror images of each other that are non-superposable , much as one's left and right hands are the same except for opposite orientation. It can be clearly understood if you try to place your hands one over the other without...

s could easily be separated with HPLC. The synthesis of epibatidine has brought a way to bypass the limitations of the substance's scarcity. It has proven to be quite productive, with a yield of about 40%.
There have also been successful attempts to create epibatidine-like molecules, like ABT-418 and epiboxidine. This was done in order to find an analgesic with less adverse effects.

Structure and reactivity


Epibatidine’s structure is very similar to that of nicotine
Nicotine
Nicotine is an alkaloid found in the nightshade family of plants that constitutes approximately 0.6–3.0% of the dry weight of tobacco, with biosynthesis taking place in the roots and accumulation occurring in the leaves...

. After this discovery in 1992, epibatidine had been proven to have similar bioactivation as its structural nephew
Nicotine
Nicotine is an alkaloid found in the nightshade family of plants that constitutes approximately 0.6–3.0% of the dry weight of tobacco, with biosynthesis taking place in the roots and accumulation occurring in the leaves...

. Other than its agonistic abilities to provide a similar antinociceptive response in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...

, epibatidine is no reactive compound. It is a hygroscopic oily substance that can act as a base
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...

, due to its nitrogenous aspects. Therefore, it can bind acid
Acid
An acid is a substance which reacts with a base. Commonly, acids can be identified as tasting sour, reacting with metals such as calcium, and bases like sodium carbonate. Aqueous acids have a pH of less than 7, where an acid of lower pH is typically stronger, and turn blue litmus paper red...

s to form salts.
Its structural resemblance to nicotine enables the substance to bind nicotinic acetylcholine receptors. However, the small difference in structure gives epibatidine a more powerful affinity to the receptors than nicotine. It acts as an acetylcholine agonist
Agonist
An agonist is a chemical that binds to a receptor of a cell and triggers a response by that cell. Agonists often mimic the action of a naturally occurring substance...

 to invoke the release of dopamine
Dopamine
Dopamine is a catecholamine neurotransmitter present in a wide variety of animals, including both vertebrates and invertebrates. In the brain, this substituted phenethylamine functions as a neurotransmitter, activating the five known types of dopamine receptors—D1, D2, D3, D4, and D5—and their...

 and noradrenaline. Ultimately, these components will cause analgesia at low doses.

Epibatidine can also bind to muscarinic acetylcholine receptors as an antagonist
Receptor antagonist
A receptor antagonist is a type of receptor ligand or drug that does not provoke a biological response itself upon binding to a receptor, but blocks or dampens agonist-mediated responses...

. This is, however, at a much lower affinity than to the nicotinic acetylcholine receptor. Binding to these receptors only occurs at a higher than therapeutic internal concentration: 8-16 µg kg−1. By disabling these postsynaptic neurons, paralysis will occur. When the nerves to vital organs get disrupted by epibatidine, death will certainly follow.

Mechanism of action



Epibatidine has two mechanisms of action: it can bind either the nicotinic acetylcholine receptors (nAChR) or the muscarinic acetylcholine receptors (mAChR)
  • The analgesic
    Analgesic
    An analgesic is any member of the group of drugs used to relieve pain . The word analgesic derives from Greek an- and algos ....

     property of epibatidine works by binding nicotinic acetylcholine receptor (nAChR) binding sites.

Nicotinic acetylcholine receptors are positioned in the postsynaptic membrane of nerve cells. They propagate neurotransmission in the central
Central nervous system
The central nervous system is the part of the nervous system that integrates the information that it receives from, and coordinates the activity of, all parts of the bodies of bilaterian animals—that is, all multicellular animals except sponges and radially symmetric animals such as jellyfish...

 and peripheral nervous system
Peripheral nervous system
The peripheral nervous system consists of the nerves and ganglia outside of the brain and spinal cord. The main function of the PNS is to connect the central nervous system to the limbs and organs. Unlike the CNS, the PNS is not protected by the bone of spine and skull, or by the blood–brain...

. When neurotransmitters bind to these receptors the ion channel opens allowing Na+ and Ca2+ ions to move across the membrane. This depolarizes the post synaptic membrane inducing an action potential that will propagate the signal. This signal will ultimately induce a release of several substances among which are dopamine
Dopamine
Dopamine is a catecholamine neurotransmitter present in a wide variety of animals, including both vertebrates and invertebrates. In the brain, this substituted phenethylamine functions as a neurotransmitter, activating the five known types of dopamine receptors—D1, D2, D3, D4, and D5—and their...

 and norepinephrine
Norepinephrine
Norepinephrine is the US name for noradrenaline , a catecholamine with multiple roles including as a hormone and a neurotransmitter...

, resulting in an antinociceptive effect on the organism.
The usual neurotransmitter for nAChR is acetylcholine yet other substances like epibatidine and nicotine
Nicotine
Nicotine is an alkaloid found in the nightshade family of plants that constitutes approximately 0.6–3.0% of the dry weight of tobacco, with biosynthesis taking place in the roots and accumulation occurring in the leaves...

 are able to bind the receptor as well and induce a similar, if not identical, response. Epibatidine has an extremely high affinity for nAChR’s and will induce a response at concentrations of ~10 µM. This is a 1000x lower concentration when compared to a nicotine induced response.
  • The paralytic property of epibatidine works by binding muscarinic acetylcholine receptors (mAChR)

These receptors are G-protein coupled of five subtypes. M2 and M4 are coupled to an inhibitory protein which impedes the functioning of adenylyl cyclase. This enzyme catalyses the reaction of ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...

 into cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...

, which is an important second messenger in a cell.
M1, M3 and M5 are coupled to a Gq-protein, which will activate the phosphatidylinositol 3-kinases
Phosphoinositide 3-kinase
Phosphatidylinositol 3-kinases are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. In response to lipopolysaccharide, PI3K phosphorylates p65, inducing...

 (PI3K). These enzymes will, when activated, catalyze the reaction of Phosphatidylinositol 4,5-bisphosphate (PIP2)into diacylglycerol
Diglyceride
A diglyceride, or a diacylglycerol , is a glyceride consisting of two fatty acid chains covalently bonded to a glycerol molecule through ester linkages....

 (DAG) and inositol-1,4,5-triphosphate (IP3). These second messengers can affect several processes in the cell, such as (sarco) endoplasmic reticulum calcium ATP-ase (SERCA).

Low doses of epibatidine will only affect the nAChRs, due to a higher affinity to nAChRs than to mAChRs. Higher doses, however, will cause epibatidine to bind to the mAChRs and cause the paralytic effects.

Metabolism


Oddly enough, in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...

 studies seem to suggest that epibatidine is hardly, if at all, metabolized in the human body.
Also there is currently little information on the path of clearance. Maximum concentration in the brain is about 30 minutes after injection and epibatidine is still present after 4 hours. This shows that the clearance has a low rate.
Speculations can be made as to how the metabolism of epibatidine works, since the large similarities with nicotine.

Toxicity


Epibatidine has several toxic consequences. Empirically proven effects include splanchnic
Splanchnic nerves
The splanchnic nerves are paired nerves that contribute to the innervation of the viscera, carrying fibers of the autonomic nervous system as well as sensory fibers from the organs...

 sympathetic
Sympathetic nervous system
The sympathetic nervous system is one of the three parts of the autonomic nervous system, along with the enteric and parasympathetic systems. Its general action is to mobilize the body's nervous system fight-or-flight response...

 nerve discharge and increased arterial pressure. The nerve discharge effects can cause antinociception partially mediated by agonism of central nicotinic acetylcholine receptors at low doses of epibatidine; 5 µg kg−1. At higher doses, however, epibatidine will cause paralysis and loss of consciousness, coma and eventually death. The median lethal dose  (LD50) of epibatidine lies between 1,46 µg kg−1 and 13,98 µg kg−1. This makes epibatidine somewhat more toxic than dioxin (with an average LD50 of 22,8 µg kg−1). Due to the little difference between its toxic concentration and antinociceptive concentration, its therapeutic uses are very limited.

Epibatidine is a rare compound in nature and since it appears to serve no useful medical uses, it will not be produced in the near future. Epibatidine does not pose any threat to the environment whatsoever.

Efficacy and side effects


Epibatidine has a high analgesic potency, as stated above. Studies show it has a potency at least 200 times that of morphine
Morphine
Morphine is a potent opiate analgesic medication and is considered to be the prototypical opioid. It was first isolated in 1804 by Friedrich Sertürner, first distributed by same in 1817, and first commercially sold by Merck in 1827, which at the time was a single small chemists' shop. It was more...

. As the compound was not addictive nor did it suffer the effects of habituation, it was very promising to replace morphine as a painkiller. Unfortunately for its therapeutic uses, the therapeutic concentration is very close to the toxic concentration. This means that even at a therapeutic dose (5 µg kg−1) some of the epibatidine might bind the muscarinic acetylcholine receptors and cause adverse effects, such as hypertension
Hypertension
Hypertension or high blood pressure is a cardiac chronic medical condition in which the systemic arterial blood pressure is elevated. What that means is that the heart is having to work harder than it should to pump the blood around the body. Blood pressure involves two measurements, systolic and...

, bradycardia
Bradycardia
Bradycardia , in the context of adult medicine, is the resting heart rate of under 60 beats per minute, though it is seldom symptomatic until the rate drops below 50 beat/min. It may cause cardiac arrest in some patients, because those with bradycardia may not be pumping enough oxygen to their heart...

 and muscular paresis
Paresis
Paresis is a condition typified by partial loss of voluntary movement or by impaired movement. When used without qualifiers, it usually refers to the limbs, but it also can be used to describe the muscles of the eyes , the stomach , and also the vocal cords...

. There has been a significant amount of research towards creation of a derivative of epibatidine characterized by reduced toxicity, whilst retaining the powerful analgesic effect. To date these efforts remain unsuccessful, but epibatidine and its relatives may still have a role in pain management in the future.

Effects on animals


In research on mice, administration of doses over 5 μg kg-1 of epibatidine caused a dose-dependent paralyzing effect on the organism. With doses over 5 μg kg-1, symptoms included hypertension
Hypertension
Hypertension or high blood pressure is a cardiac chronic medical condition in which the systemic arterial blood pressure is elevated. What that means is that the heart is having to work harder than it should to pump the blood around the body. Blood pressure involves two measurements, systolic and...

 (increased blood pressure), paralysis in the respiratory system, seizures, and, ultimately, death. The symptoms do, however, change drastically when lower doses are given. Mice became resistant to pain and heat with none of the negative effects of higher doses. Compared to the gold standard in pain management, morphine
Morphine
Morphine is a potent opiate analgesic medication and is considered to be the prototypical opioid. It was first isolated in 1804 by Friedrich Sertürner, first distributed by same in 1817, and first commercially sold by Merck in 1827, which at the time was a single small chemists' shop. It was more...

, epibatidine needed only 2.5 μg kg−1 to initiate a pain-relieving effect whilst the same effect required approximately 10 mg kg−1 of morphine. Currently, only rudimentary research into epibatidine's effects has yet been performed; the drug has been administered only to rodents for analysis at this time.

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