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Dear group,
I am working on a project of midazolam right now. 70%(fmet) of midazolam (MDZ) can metabolized to
1-hydroxymidazolam (1-OH-MDZ) by CYP3A. In the literatures, if MDZ is given by iv, after adding
CYP3A inhibitors orally, MDZ AUC and 1-OH-MDZ AUC both goes up.
We know that after iv administration, AUC of metabolite=Dose of MDZ*fmet/CLtot of metabolite. If we
know the inhibitor has no effect on CLtot of metabolite or other metabolism pathways (CLother of
MDZ), fmet should go down, CLtot of metabolite should not change, so the AUC of metabolite should
also go down. But in all the literatures, 1-OH-MDZ AUC goes up.
Can anyone explain this to me? I am really confused here. Thank you very much!
Best,
Mengyao
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Mengyao-
When tying to understand metabolite AUC (AUCm), it is important to always view it relative to parent
AUC (AUCp) i.e. AUCm/AUCp should be your measure of interest, not absolute AUCm.
AUCm/AUCp is ultimately the ratio of metabolite formation clearance (CLf) to metabolite elimination
clearance (CLm):
AUCm/AUCp = fm*CLp/CLm = CLf/CLm
When CYP3A4-mediated midazolam elimination (i.e. hydroxymidazolam formation) is inhibited, the AUCp
will increase and the AUCm may increase as well because of increased parent exposure, but AUCm/AUCp
will decrease because Clf decreased (assuming the inhibitor has no effect on Clm).
A quick PubMed search will show that often 1-hydroxymidazolam AUCm increases after CYP3A4 inhibitor
administration, but not as much as midazolam AUCp, ultimately AUCm/AUCp decreases (and hence Clf
decreases).
Please check the 1-hydroxymidazolam AUCm/AUCp for your specific project and let us know if it
decreases/increases/remains the same. If it doesn't decrease, we can then talk about the possibility
of treatment effects on metabolite elimination (Clm). I look forward to hearing your response…
-Justin Lutz
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Mingyao why do you think that the inhibitor has no effect on CLtot of the metabolite? Most of the
time primary metabolites are substrates of the same enzymes and transporters as the parent drug.
When this happens the AUC of the metabolite goes the same direction as the parent in drug
interaction studies. In fact, when the AUC of the metabolite goes the same direction as the parent
in a drug interaction study it is diagnostic that the metabolite is a substrate for the same enzymes
and transporters as the parent. fmet does go down, but you cannot tell it by looking at AUCs. We
have pointed this out in a number of our papers, and even have it in the title of our 2009 glyburide
study. Elucidating Rifampin’s Inducing and Inhibiting Effects on Glyburide Pharmacokinetics and
Blood Glucose in Healthy Volunteers: Unmasking the Differential Effects of Enzyme Induction and
Transporter Inhibition for a Drug and Its Primary Metabolite. H.X. Zhang, Y. Huang, L.A. Frassetto
and L.Z. Benet. Clin. Pharmacol. Ther. 85, 78-85 (2009).
Leslie Z. Benet, Ph.D.
Professor
Department of Bioengineering & Therapeutic Sciences
Schools of Pharmacy & Medicine
University of California San Francisco
533 Parnassus Avenue, Room U-68
San Francisco, CA 94143-0912
Email: leslie.benet.-at-.ucsf.edu
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Dr. Benet,
Thank you for your response. But in the case of midazolam, its primary metabolite 1-OH-MDZ is
metabolized by UGT to conjugated 1-OH-MDZ (account for 60%-70% of MDZ dose) [1], instead of CYP3A.
All the inhibitors in the studies (saquinavir, voriconazole, fluoconazole, grapefruit juice) are not
UGT inhibitors or substrate at clinical relevant concentration, except for saquinavir. So I think
CLtot of metabolite will not change in this case.
[1] Zhu B. et al. Characterization of 1'-Hydroxymidazolam glucuronidation in Human Liver
Microsomes.DMD. 2007, 36: 331-338.
Best,
Mengyao
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Justin,
Thank you for the explanation. But the equation AUCm/AUCp=fm*CLp/CLm is based on the equations that
AUCm=Dose*fm/CLm (1)
AUCp=Dose/CLp (2)
When we look at equation (1), Dose is not changed, assuming CLm is not changed, the only explanation
AUCm goes up is that fm goes up. The increased parent exposure has no effect on AUCm here, I think.
Best,
Mengyao
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Thanks Mengyao. I answered too quickly.
Here is what I should have said:
Mingyao why do you think that the inhibitor has no effect on CLtot of the metabolite? Most of the
time primary metabolites are substrates of the same enzymes and transporters as the parent drug.
When this happens the AUC of the metabolite goes the same direction as the parent in drug
interaction studies. In fact, when the AUC of the metabolite goes the same direction as the parent
in a drug interaction study it is diagnostic that the metabolite is a substrate for enzymes and
transporters AT THE SAME METABOLIC SITE as the parent. fmet does go down, but you cannot tell it by
looking at AUCs. We have pointed this out in a number of our papers, and even have it in the title
of our 2009 glyburide study. Elucidating Rifampin’s Inducing and Inhibiting Effects on Glyburide
Pharmacokinetics and Blood Glucose in Healthy Volunteers: Unmasking the Differential Effects of
Enzyme Induction and Transporter Inhibition for a Drug and Its Primary Metabolite. H.X. Zhang, Y.
Huang, L.A. Frassetto and L.Z. Benet. Clin. Pharmacol. Ther. 85, 78-85 (2009).
I still believe that "most of the time" primary metabolites are substrates of the same enzymes and
transporters as the parent drug. However, in the case you asked about, you are correct and this is
not true. The 1-hydroxymidazolam is primarily glucuronidated and
eliminated in the urine as shown by Zhu et al. in MICROSOMES. If they had run the study in
hepatocytes over 10-60 min, as they did in microsomes, they would have seen little glucuronidation.
This is because hepatic metabolism is always affected by permeability into the hepatocyte,
permeability out of the hepatocytes into the bile via the apical membrane and permeability back into
the blood via the basolateral membrane, in addition to metabolism. Phase 1 metabolites are more
polar than the parent compound (the major purpose of metabolism) and their permeability into the
hepatocyte will be much less than the parent drug. When a drug is susceptible to sequential
metabolism, as for midazolam, a good portion of the primary metabolite will be sequentially
metabolized to the glucuronide, but a small portion of the primary metabolite will be kicked back
into the blood and then this metabolite has much more difficulty getting into the hepatocyte than
the parent compound. (This is why dosing a metabolite usually provides no useful information about
the metabolite kinetics when the parent drug is dosed.) Thus your metabolic inhibitor may not be
inhibiting the Phase 2 enzyme, here glucuronosyltransferase, but appears to be inhibiting the access
of the primary metabolite to the Phase 2 enzyme, possibly both within the hepatocyte and by making a
metabolite that has difficulty getting back into the hepatocyte, and thus changing CLtot of the
metabolite. When the access of the metabolite to the Phase 2 enzyme is limited, then the Phase 1
enzyme will accumulate as the parent drug. There is another issue. Midazolam is a BDDCS Class 1
drug and should not be affected by transporters in the liver, but there is a good chance that
1-hydroxymidazolam could be a BDDCS Class 2 drug and could be affected by transporters.
Your question is a very good one, and there has been little work examining the potential pathways I
have discussed to explain the answer. Our limited work in this area has concentrated on primary
metabolites that are substrates for the same enzymes and transporters as the parent (atorvastatin,
glyburide, cyclosporine, tacrolimus).
Les
Leslie Z. Benet, Ph.D.
Professor
Department of Bioengineering & Therapeutic Sciences
Schools of Pharmacy & Medicine
University of California San Francisco
533 Parnassus Avenue, Room U-68
San Francisco, CA 94143-0912
Email: leslie.benet.-at-.ucsf.edu
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Mengyao-
I agree with Les in that if Clm is rate limited by hepatic influx, then assuming that there is no
change in Clm because your precipitants do not inhibit UGT may not be valid. P450-transporter
interplay is certainly an important factor to consider.
Another possible explanation can arise from considering what is AUCm and fm (after IV bolus dosing):
AUCm=fm*D/Clm
fm=Clf/Clp
If there are metabolite clearance pathways other than 1-hydroxy Clf (let’s call this other pathway
Clo), then:
Clp=Clf+Clo
fm=Clf/(Clf+Clo)
AUCm=Clf*D/(Clf+Clo)*Clm
If Clf decreases to a lesser extent than Clo, fm will increase and hence AUCm will increase. So your
thinking that fm must increase is possibly true, just remember that fm is a function of all
clearance pathways in the system. Ultimately an increase in both AUCp and AUCm can be diagnostic of
influx transporter inhibition and/or “other metabolic pathway” inhibition.
If CYP3A4 forms other metabolites (such as 4-hydroxymidazolam) and this pathway is also inhibited,
the relative extent of inhibition for each metabolic pathway becomes important to consider. Because
of allosterism, it shouldn’t be assumed that your precipitant will inhibit both formation clearances
equally. Yang et al. (Clin Pharmacol Ther 2012) demonstrated that 1-hydroxy is more susceptible to
in vivo 3A4 inhibition by fluconazole than 4-hydroxy, but this relative susceptibility is expected
to be inhibitor-dependent due to the allosteric nature of the interaction.
One important aspect to the above equation is that Clf and Clo have to be comparable in order for
Clo inhibition to significantly increase AUCm. This is probably unlikely with 4- and
1-hydroxymidazolam, yet increased 1-hydroxy AUCm is well documented. I agree with Les that you have
indeed pointed out a perplexing in vivo phenomenon.
Justin Lutz, PharmD PhD
Research Investigator II
Department of Biotransformations
Bristol-Myers Squibb - Princeton, NJ
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