Hi Claire, depending on the molecular structure of your compound, radiochemistry could provide a pathway exploring this in great detail.

You will need to experienced radio pharmacy/radiochemistry folks to discuss pathways to do this. If there is provision to label your molecule without altering its structure or at sites known not to change/influence its actions, you have a good chance.

Once there, you may apply PET, SPECT or radio HPLC/TLC to check for metabolism with an enormous sensitivity. Though it is not that easy to structure-ID the metabolite. Just recognition by comparing separation.

Otherwise you may try classical analytical approaches, e.g. Nano-UPLC-MS of samples from the brain of the treated animals.

Here the limits will be on the sensitivity side of things.

Hi Marko, thanks very much for your answer. Another question though! How do I find out if there is provision to label my molecule without altering its structure? Is this something I can find out before bothering the radiochemists or is this a trial and error thing?

I do have access to PET, SPECT and HPLC within the dept, although as a cell biologist, this is slightly out of my comfort zone and would like not to ask completely idiot questions! Thanks again, Claire

Similarly to what said above, if you do not want to go into radiolabelling you could always mark your molecule with a fluorophore and then simply proceed with IHC.

Still, you should do some tests to check that the molecule is still bioactive.

As for predicting whether the fluorophore/radioligand will interphere, I think the chemist can do some predictions if they have access to some structural data on how the molecule binds to its receptor. However, even with that, I think there is still some "trial and error" involved, and surely in any case you need to test your marked molecule afterwards with some biological assay.

Hi Claire,

A few thoughts for your questions:

1) I would like to know whether the drug itself gets into the brain at all - PET/SPECT imaging would work for this type of purpose, but you need to labeled your molecule with either PET/SPECT isotope; if you have a H-3/P-32 labeled molecule, you can also do tissue extraction and scintillation counting;

2) whether it is a metabolite or secondary effect that is protecting? Is my only option trying radiochemistry? - incubate your compound in the homogenized brain tissue and extract the compounds/metabolite and do HPLC-MS to figure this out.

Good luck!

Hi Claire,

I would suggest to turn to a carbon 11 labeling and PET imaging with a biodistribution study; you have all the skills and knowledge and equipment around you at King's college (would be worth to speak with Pr. Tony Gee). a 14C, 13C or even Tritium labeling could also be used associated with HPLC and Mass spectroscopy of brain/organ extracts. i wouldn't recommend the use of SPECT except if your compound already contain Iodine (then you could use 123I).

Of course it means to do a new synthesis of the compound.

If your chemical contains fluorine then consider a fluorine-18 labeling (sometimes more easy than 11C and more appropriate if a longer distribution time is needed to cross the blood brain barrier).

For metabolite this is always a hard task, the best is to try brain extracts associated with HPLC and mass spectroscopy with or without a radioactive probe.

good Luck

there is little more add. You need to take your compound's formula and synthesis to a radiochemist. They can advice you on potential pathways.

11C has only a half lifetime of 20min. you can analzye it for a few half life times. Nice thing is that if you compound comes with methylated groups, it is a natural fit.

Talk to a chemist on this one with your data, formula etc. We have hit about the limit on how to go forward without knowing more specifics.

If you employ radio methods, you will discover how powerful they are as a tool. It is almost magic what you can do compared to traditional methods for tracing compounds due to the extreme sensitivity and ease of detection.

There is a lot of infrastructure required, but there are experts and labs around to support you.

Best

Marko

Hi All, thanks so much for all the useful info delivered so rapidly, and I certainly have a clearer idea of how to go ahead. I think I will explore the possibility of 11C as the compound does have methyl groups.

Patrice, thanks again. I will definitely get in touch with Tony - in fact he visited the lab yesterday, coincidentally. Do you know him?

Hi Claire,

Tony Gee is famous in radiochemistry, i had the pleasure top meet him last year, he often came to Strasbourg/France for Radiochemistry courses and lecturers.

11C labeling can be a good solution depending on the structure of your compounds, especially if you have N-Methyl, O-methyl or S-methyl group. but beware of the so short period...it can impaired the biological study (too long biodistribution time versus half life of 11C ). Is your compound a peptide ?

Best regards

Hi Patrice, didn't realise that Tony was a legend!

Have tried to attach a structure of the compound below, although not sure if it's worked. Any thoughts you may have, appreciated.

Hi,

You need to set up a biodistribution study and try to find out by using assays. I would recommend radiolabelling of a compounds which is known to be involved in the pathway. It is a long road but it is worth it if you really want to know how it is metabolized.

Another option is to send your request to a pharmacology laboratory and ask them to do the job for you (cost a lot but it save you time).

Contact Tony Gee is the best thing you can do, since he is in my opinion the world best radiochemist.

Wish you all the best

Vic

Hi Claire,

I am not that skilled in 11C chemistry but according to the structure you attached above, a labeling will be really difficult without modifying your initial structure !

if the compound having a SO2-NHCH3 is also active then it would be more easy to synthesize the 11C derivative. but this mean to redo the activity test on such a compounds.

best regards

Thanks Patrice. I am completely at the limit of my chemistry unfortunately and couldn't predict what effect modulating the structure would have on the protection (and the prospect of redoing all the animal expts is slightly daunting!). However I have an in vitro assay of protection that I could start with if structural alteration is the only way to get any kind of preclinical study off the ground.. But I have an appt with Tony Gee in a fortnight so will put your suggestion to him. Thanks again for your help.

Claire, in this case I suggest to go for S35 labeling. Half life of some 87 days and a beta emitter. This shoulf be easy to incorporate. Disadvantage is that you need to slice your samples and put them on a radiographic scanner.

Hi Marko, that's also something we could consider as we definitely have the facilities to do it. Thanks very much for your help.

Mentabolites can be separated by radio-HPLC or TLC.

Identification of metabolites is a tad more complicated.

Though the HPLC/TLC will tell you that you have either your compound in the brain or a mix or just a metabolite. If you can see the tracer in the radiography, you can also see it on a TLC plate or the radio-hplc.