Mutasim
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Jordan,
2019-09-23 12:40

Posting: # 20641
Views: 900
 

 Ezetimibe BE [Regulatives / Guidelines]

Dear Members,
Would you please advice on interpretation of the requirements for Ezetimibe BE according to the last guidance by EMA, in particular the statement
"Main pharmacokinetic variables: AUC0-72h, Cmax
Background/justification: On total (parent + glucuronide metabolite together)
Does it mean measuring each one alone? or the total as in FDA guidance? and accordingly will the CI be for each one alone or both unconjugated and total or only total?


Edit: Guideline linked. [Helmut]
Helmut
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2019-09-23 13:36

@ Mutasim
Posting: # 20642
Views: 820
 

 Ezetimibe BE

Hi Mutasim,

» Does it mean measuring each one alone? or the total as in FDA guidance?

The glucuronide is much more hydrophilic than the parent. No way to extract both together in sample preparation (not to speak about having them in the same chromatographic run – unless you apply an aggressive gradient elution). Hence, use glucuronidase. This gives you the total.

» CI be for each one alone or both unconjugated and total or only total?

Total.

Cheers,
Helmut Schütz
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Helmut
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2019-09-25 09:52

@ Mutasim
Posting: # 20647
Views: 702
 

 Confusing guidance

Hi Mutasim,

reading the guidance again I understand your confusion.

On top of page 3 we have

Analyte:parentmetaboliteboth
Background: Ezetimibe undergoes extensive pre-systemic metabolism; ezetimibe-glucuronide is the major active metabolite. Because of extensive hepatic recirculation, the exposure to ezetimibe is less representative to evaluate absorption.

And then

Bioequivalence assessment: Background/justification: On total (parent + glucuronide metabolite together)

(my emphases)

Analyte:both seemingly tells us to measure them separately but the remainder tells us that only the total should be assessed for BE. So why all that fuzz?

I still think that cleaving by glucuronidase and analyzing ezetimibe is the way to go. What I wrote above was based on extraction techniques (SPE, LLE). If you opt for protein precipation it could work but as a hydrophilic compound it might well be that some part of the glucuronide gets trapped in the precipitate. I also believe that you need two chromatographic conditions. Furthermore, glucuronides are known for back-conversion in the ion-source. Hence, what you might get are wrong values for both the parent and the metabolite but the sum will be correct (assuming that nothing of the glucuronide gets trapped – what I doubt). Hence, I would keep it simple and go for cleavage and have just one run.
If the EMA really want both separatelly IMHO, it would have been better to state in the GL:

Analyte:parentmetaboliteboth


Out of curiosity: Since dealing with glucuronides was the topic of my first paper* – which stationary & mobile phase are you using?


  • Mascher H, Nitsche V, Schütz H. Separation, isolation and identification of optical isomers of 1,4-benzodiazepine glucuronides from biological fluids by reversed-phase high-performance liquid chromatography. J Chr B. 1984; 231–9. doi:10.1016/S0378-4347(00)80885-3.

Cheers,
Helmut Schütz
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nobody
nothing

2019-09-25 11:36

@ Helmut
Posting: # 20648
Views: 687
 

 Confusing guidance

imho the authority wants you to be AWARE that there is a (reversible? during plasma preparation? storage? under chromatographic conditions?) glucuronidation relevant for the compound. Did you have a look at the PAR or alike for PK details?

Kindest regards, nobody
Helmut
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2019-09-28 10:11

@ Mutasim
Posting: # 20650
Views: 596
 

 Ezetimibe final guidance

Hi Mutasim & all,

yesterday at BioBridges Paola Coppola (MHRA, PKWP observer) presented the state of affairs. The ezetimibe guidance is finalized and will be published soon. No changes to the draft. You are right and I was wrong. One should indeed measure both ezetimibe and its glucuronide. BE should be based on their sum* only.

I still think that’s stupid. Both methods have errors (inaccurate, imprecise). If you evaluate the sum of concentrations, errors will sum up as well. Given, the concentrations of the glucuronide are two orders of magnitude higher than the parent’s and hence, the impact of the latter is negligible.
At the BioInternational 1994 in Munich there was a nice distinction between “Need to know” and “Nice to know”. I don’t like the idea that something which is only nice to know (i.e., not used in the assessment for BE) is mandatory in a guidance. :thumb down:


  • Bioequivalence assessment: Background/justification: On total (parent + glucuronide metabolite together)
    (my emphases)
    Cave: Apples & oranges. Total should be expressed as ezetimibe after correction for the molecular masses (Mezetimibe 409.4 g·mol–1, Mezetimibe glucuronide 585.5 g·mol–1).

Cheers,
Helmut Schütz
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Ohlbe
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France,
2019-09-30 09:03

@ Helmut
Posting: # 20655
Views: 498
 

 Ezetimibe final guidance

Dear Helmut,

» One should indeed measure both ezetimibe and its glucuronide. BE should be based on their sum only.

» Both methods have errors (inaccurate, imprecise). If you evaluate the sum of concentrations, errors will sum up as well.

Well... It depends how you measure the glucuronide. If you have a direct method (which is a bit of a challenge), that's true, but the fact that

» the concentrations of the glucuronide are two orders of magnitude higher than the parent’s

will limit the impact.

But if you directly measure total (after deconjugation), and then calculate the glucuronide by substracting unconjugated from total, you won't have this problem. The increased error will affect the glucuronide, but you don't use these concentrations for the determination of bioequivalence.

Wouldn't this be acceptable ?

Regards
Ohlbe
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2019-09-30 13:33

@ Ohlbe
Posting: # 20658
Views: 470
 

 Ezetimibe final guidance

Dear Ohlbe,

I agree with your points about error, etc.

» But if you directly measure total (after deconjugation), and then calculate the glucuronide by substracting unconjugated from total, you won't have this problem. The increased error will affect the glucuronide, but you don't use these concentrations for the determination of bioequivalence.
»
» Wouldn't this be acceptable ?

I don’t know what is acceptable for the agencies. For me, absolutely. I still don’t get the point why one has to present the aglycone at all. Heck, it is not used in the BE assessment.

Cheers,
Helmut Schütz
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ElMaestro
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Belgium?,
2019-09-30 11:12

@ Mutasim
Posting: # 20656
Views: 489
 

 Ezetimibe BE

"The applicant should document that the enzymatic de-conjugation can be considered quantitative under assay conditions"

:-D:vomit:

I could be wrong, but...
Best regards,
ElMaestro
Helmut
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2019-09-30 13:26

@ ElMaestro
Posting: # 20657
Views: 471
 

 Ezetimibe BE

Hi ElMaestro,

» "The applicant should document that the enzymatic de-conjugation can be considered quantitative under assay conditions"
»
» :-D:vomit:

That would be a valid request. Has to be determined in method development & validation. No big deal, since β-glucuronidase is used for decades.

Cheers,
Helmut Schütz
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