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Improving Codeine conversion via enzyme manipulation

Megalum

Megalum

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Jul 10, 2010
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101


codeinemorphine-pk.png


*source https://www.pharmgkb.org/pathway/PA146123006#

The studies so far show mixed results on UGT2B7 effectiveness. So yes, this is a probably a pipe-dream thought... but, you know... drugs.

I know everyone would love a successful means to have more Codeine -> Morphine conversion rate higher than the avg 10%~
People have looked ALL through CYP2D6 and there isn't much there as of yet. Theoretically, inhibiting UGT2B7 could have some real potential.
For one, it would slow the conversion of the relatively useless codeine-6-glucuronide,
UGT2B7 catalyzed morphine glucuronidation at the 3- and 6-hydroxy positions and also mediated the formation of codeine-6-glucuronide from codeine.
Click to expand...

*sourcehttps://www.pharmgkb.org/pmid/9010622

Now, I realize this would also lower the amount of the 3 and 6 forms of metabolized morphine, but that seems a small sacrifice for the extra CYP2D6 pathway being forced to metabolize more codeine.

That being said, I need help researching a list of possible candidates to inhibit UGT2B7 -- the most likely source I've seen so far is Diclofenac, here is some data regarding the aforementioned.
http://www.cyprotex.com/useruploads/UGT-inhibition-fig3-5.png

For record, they used Naloxone as the substrate to test diclofenac's potency with regard to inhibitory effect.

Any comments, discussion, please help me crack the code :)
 
Well i have been prescribed Diclofenac along with Codeine for a few injuries over the years and it didn't seem to add to the effect of Codeine. All it really did was help the inflammation thus it added to the analgesic effects abit but every anti-inflammatory does that. It's a pretty common combo for alot of injuries so i doubt there's anything to that.

Id say your best bet would be white grapefruit juice. I find that drinking it about a hour before taking Codeine helps abit anyway.
 
There's no way to force it to the morphine by inhibiting downstream, it could just as easily form norcodeine instead. Your best bet is to induce 2D6/7 which is also part of hydrocodone being formed into hydromorphone. This has been tested and does work, I cannot remember the chemical off the top of my head, but there is a laundry list of them, some more effective than others, some with less side effects also. I think it was a first generation antihistamine or something like that. I also believe carbamazapine in general induces CYP450 enzymes too. If you could block 3A4 and induce 2D6 at the same time you could dramatically flip the balance. Grapefruit juice is not going to increase morphine concentrations, just delay codeine becoming norcodeine.
 
PINKoPANaTHER said:
Grapefruit juice is not going to increase morphine concentrations, just delay codeine becoming norcodeine.
Click to expand...

And thereby increase the codeine to morphine conversion. And if morphine has a slower elimination rate than codeine's O-demethylation, then it will increase morphine concentrations. I don't think inhibiting codeine glucuronidation is going to help you a lot, because C6G is believed to be active as well, at least some papers suggest that, like this one for example. However, inhibiting morphine 3-glucuronidation should be useful. Still, I think your best bet is inhibiting CYP3A4 to stop N-demethylation or inducing 2D6.

E: could someone explain why codeine is practically inactive while C6G is supposedly active. Isn't a hydrogen bond donor in the 3rd position required for appropriate binding? Could the bulky glucuronide with its hydroxyls provide a HBD in that vicinity instead?
 
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It doesn't make sense that C6G is active. Maybe C3G could be active because the glucuronyl moiety can donate an H-bond (H bond donation is required at carbon 3, but not at carbon 6). Where is the paper that C6G is active? Maybe C6G is metabolised to M6G which is what provides the activity.
 
morphine 6 glucuronide is active, i haven't heard before that C6G is active but the analogy could make sense
 
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another possible explanation for G6C activity could be that the glucuronic ring interacts with a different site increasing affinity, since the lowest energy conformation will have the glucuronic acid orientated in the opposite sense respect to the morphinan ring
 
Another issue I have with the activity of these glucuronic acid derivatives is that they are generated in the liver and have absolutely poor lipophilicity so I don't see how they'd be able to get into the brain with any appreciable concentration.
 
Ive seen papers saying M6C is indeed active but forget why and how strong the effects really are. I will dig them up.
 
Kittycat5 said:
Ive seen papers saying M6C is indeed active but forget why and how strong the effects really are. I will dig them up.
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How is it getting into the brain though? Can't be diffusion across BBB. Active transport? Maybe but why? It definitely doesn't get glucuronylated in the brain...
 
aced126 said:
It doesn't make sense that C6G is active. Maybe C3G could be active because the glucuronyl moiety can donate an H-bond (H bond donation is required at carbon 3, but not at carbon 6). Where is the paper that C6G is active? Maybe C6G is metabolised to M6G which is what provides the activity.
Click to expand...

I don't know where exactly in space the HBD needs to be, but the glucuronyl is quite bulky and if you look at the 3D structure of codeine, you can see that there may be a conformer where one of the hydroxyls is able to act as a HBD near the vicinity of 3rd position. At least that's what I proposed as a possible explanation in case C6G IS active (maybe it's not and the paper missed something, like C6G -> M6G conversion for example). If you do a quick search, you will find a couple articles exploring that.

http://onlinelibrary.wiley.com/doi/10.1016/S1090-3801(97)90103-8/abstract

http://europepmc.org/abstract/med/11092114
 
Kittycat5 said:
Ive seen papers saying M6C is indeed active but forget why and how strong the effects really are. I will dig them up.
Click to expand...
M6G is active and is very potent -- several x morphine. It is thought to act on a splice varient of MOR; it is likely that interactions with the sugar moiety compensate for the loss of the interaction with OH.
 
The 6-OH doesn't interact that much anyway; we see 6-MAM being more potent than heroin and morphine while 3-MAM being not that potent at all.
 
The 6-OH only reduces potency. Krokodil is 6-desoxy morphine. Medically it was called Permonid and was used for a decade in the 50s-60s in Switzerland because of it's brief duration. The 3 -OH can be swapped for a carboxamide.
 
I've always thought somehow tweaking the bio-synthetic pathway in Papaver somniferum from yielding codeine to yielding heterocodeine (3-methyl-morphine to 6-methyl-morphine) would be a great feat. I know that hydromorphone is found in extremely minute amounts, I know I'm veering off topic from human metabolic pathways to plant ones, but with all the cannabis breeding to yield THC and CBD content, you'd think someone would capitalize on breeding a dilaudid poppy. "Dilaudid" already sounds like a pretty flower name. ;-j
 
Nagelfar said:
I've always thought somehow tweaking the bio-synthetic pathway in Papaver somniferum from yielding codeine to yielding heterocodeine (3-methyl-morphine to 6-methyl-morphine) would be a great feat. I know that hydromorphone is found in extremely minute amounts, I know I'm veering off topic from human metabolic pathways to plant ones, but with all the cannabis breeding to yield THC and CBD content, you'd think someone would capitalize on breeding a dilaudid poppy. "Dilaudid" already sounds like a pretty flower name. ;-j
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Heterocodeine is indeed several x codeine and it further provides evidence to the key features of the MOR pharmacophore. I'm not too sure how possible it would be to make Papaver somniferum to produce this, as the key enzyme involved probably can only selectively methylate the 3-OH. Changing this would probably mean changing the amino acid sequence of the enzyme.
 
aced126 said:
Heterocodeine is indeed several x codeine and it further provides evidence to the key features of the MOR pharmacophore. I'm not too sure how possible it would be to make Papaver somniferum to produce this, as the key enzyme involved probably can only selectively methylate the 3-OH. Changing this would probably mean changing the amino acid sequence of the enzyme.
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Yes, exactly, that isn't simply a matter of breeding, or even changing the amino acid sequence. It would require designing an entirely new enzyme. Small sequence differences are useful if you want to change substrate selectivity or alter the reaction rate. But for the proposed change you need to completely rearrange the location of the residues that catalyze the methylation. It likely isn't impossible to do that but it would require substantial resources and time. Then you would have to knockout the wildtype enzyme and insert the engineered one, which could be done using CRISPR.

If someone was biosynthesizing morphine in yeast then this type of engineering might make sense (although I'm not sure it would be worth the trouple of making a new enzyme vs. just extracting morphine and then chemically modifying it). But poppies are already dirt cheap to produce and it doesn't seem like the expense of engineering them would be a good investment for the people who grow them, since they are trying to sell their product for conversion to heroin.
 
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serotonin2A said:
Yes, exactly, that isn't simply a matter of breeding, or even changing the amino acid sequence. It would require designing an entirely new enzyme. Small sequence differences are useful if you want to change substrate selectivity or alter the reaction rate. But for the proposed change you need to completely rearrange the location of the residues that catalyze the methylation. It likely isn't impossible to do that but it would require substantial resources and time. Then you would have to knockout the wildtype enzyme and insert the engineered one, which could be done using CRISPR.
Click to expand...

How would one be able to find the correct aa sequence for the new enzyme so that it catalyses the proposed reaction well? It seems like an incredibly hard task. Has it ever been done before with other enzymes?
 
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