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Input CL to enter in PK-Sim from in vitro experiments #177

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Angelefleury opened this Issue May 16, 2018 · 9 comments

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@Angelefleury
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Angelefleury commented May 16, 2018

Dear colleagues,

I some questions about the input metabolic CL to enter in PK-Sim:

  • which process type should be selected and what value should we enter when we have an intrinsic CL measured in hepatocytes from a lab, expressed in uL/min/million cells or scaled intrinsic clearance expressed as mL/min/kg?

Considering a first order process the three options available are:

1- Intrinsic clearance – (L/min).
2- In Vitro clearance – 1/min
3- In vitro metabolic rate in the presence of liver microsomes – uL/min/mg mic.protein

  • Beside, is there a way to check how it is scaled and what the resulting plasma CL is?

I thank you in advance for your help
Kind regards
Angèle

@StephanSchaller

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StephanSchaller commented May 16, 2018

Hi Angèle,

very good question! There are actually a number of inquiries to improve IVIVE workflow in PK-Sim (#101), and I think this question fits very well.

I am assuming you mean CL by metabolizing enzymes:
image

Just for your reference: you will also find the processes explained in the help menu in Chapter 15 (scroll down about 2/3 of the chapter).

Number 1 will be your best option:
It basically calculates a specific clearance from your assay in which you use a certain volume of liver cells (so you have to know the volume of cells used in the assay). You then enter for:

  • Volume (liver): the volume of cells in the assay
  • fraction intracellular (liver): 1 (as you have only used cells in your assay and not tissue)
  • Intrinsic clearance: your originally measured intrinsic clearance (in l/min). This is not the value from above that you have already normalized by "million cells" but the raw measurement.

From this, PKSim calculates a specific clearance. Then, PK-Sim does some scaling:

  1. This specific clearance is then again normalized to a default "Metabolizing Enzyme Concentration" of 1 µmol/l (CLSpec/[Enzyme], following a screenshot from MoBi) (@Yuri05 , where does this 1 µmol/l come from, and why is it not shown in the intrinsic clearance calculation as a default value?):
    image

  2. if you have defined enzymes to be expressed in you individual, it then multiplies the CLSpec/[Enzyme] with the enzyme concentration of each organ where the enzyme is expressed, resulting in your final clearance rate (µmol/min).

To obtain a value for plasma CL, you best generate a simple simulation with an IV dose only and once you have displayed the results, you can obtain the plasma CL value from the PK-Analysis which is calculated once you click the "Show PK-Analysis" button below your plot:
image

Hope this helps!
Stephan

@krinaj

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krinaj commented May 17, 2018

Hi Angèle and Stephan,

My understanding is a bit different in regards to following:

Number 1 will be your best option:
It basically calculates a specific clearance from your assay in which you use a certain volume of liver cells (so you have to know the volume of cells used in the assay). You then enter for:

Volume (liver): the volume of cells in the assay
fraction intracellular (liver): 1 (as you have only used cells in your assay and not tissue)
Intrinsic clearance: your originally measured intrinsic clearance (in l/min). This is not the value from above that you have already normalized by "million cells" but the raw measurement.
From this, PKSim calculates a specific clearance. Then, PK-Sim does some scaling:

This specific clearance is then again normalized to a default "Metabolizing Enzyme Concentration" of 1 µmol/l (CLSpec/[Enzyme], following a screenshot from MoBi) (@Yuri05 , where does this 1 µmol/l come from, and why is it not shown in the intrinsic clearance calculation as a default value?):
image

if you have defined enzymes to be expressed in you individual, it then multiplies the CLSpec/[Enzyme] with the enzyme concentration of each organ where the enzyme is expressed, resulting in your final clearance rate (µmol/min).

Actually, something like above fits better if you are selecting "In Vitro Metabolic Rate in Presence of Liver Microsomes - First Order" process, where you can directly plug in in vitro CL results in uL/min/mg protein format. However, for "Intrinsic Clearance - First Order", liver volume is pre-populated as 2.36 L. So, my understanding is that your input for "Intrinsic Clearance" should be scaled to whole liver. So, if you have in vitro clearance from microsomes assay and have first order clearance in let's say μL/min/mg protein format, you have to scale it to ul/min per whole human liver volume. Standard scaling factors are 39-53 mg microsomal protein/gm liver (MPPGL) and human liver 25.7 gm liver/kg body weight.
Please correct me if I am wrong.

Krina

@msevestre

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msevestre commented May 20, 2018

@Angelefleury
I agree with @krinaj . Using In Vitro Metabolic Rate in Presence of Liver Microsomes - First Order is probably what you are looking for based on your description

@StephanSchaller

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StephanSchaller commented May 20, 2018

I guess it depends on your info available for scaling...

as you only have uL/min/million cells, for

  1. you need volume/cell and for
  2. you need mg mic.protein/cell
@krinaj

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krinaj commented May 20, 2018

which process type should be selected and what value should we enter when we have an intrinsic CL measured in hepatocytes from a lab, expressed in uL/min/million cells or scaled intrinsic clearance expressed as mL/min/kg?

Sorry, I did not read original question thoroughly earlier. It seems like you have hepatocytes data and not microsomal enzyme assay. It also seems like you have already scaled from uL/min/million cell to mL/min/kg. I believe instead of scaling to per kg, you can just scale to average human liver weight, and then add it in Intrinsic clearance - first order process.

So, CL in uL/min/million cells * hepatocytes per gram liver (HPGL) * liver weight (gm) in average human

HPGL = 139e+6
liver weight in average human = 1561 gm

@tobiasK2001

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tobiasK2001 commented May 22, 2018

Dear Angèle,

from the range aof answers you see it is not an easy question indeed. I hope not to confuse you totally, if I give my two pence here with a somewhat different opinion than the other gentlemen.
There are many options in PK-sim to input iv vitro CL, thus we have to be sure what you have and what you want to do:
From your question: "intrinsic CL measured in hepatocytes from a lab, expressed in uL/min/million cells or scaled intrinsic clearance expressed as mL/min/kg?", it seems to me that you have an hepatocyte stabilitiy experiment and you don't want to assigen your CL to an specific enzyme.
If you want to do an IVIVE based on a total hepatocyte CL you have to input it as "Total Hepatic Clearance" in your compound.
Unfortunatly an input in "uL/min/million cells" is not possible here.
You might imput is as "Liver Plasma Clearance" here
grafik
But be aware that the value provided by your lab expressed as ml/min/kg is most likely already a converted intrinsic Cl to a plasma CL. I guess they used the well stirred liver model and all known scaling factors. PK-sim will do the same and will recalculate from the plasma CL input using appropriate scaling factors and a well stirred liver like formula a specific intrinsic liver clearance from your input. Scaling facors are inbuild so you just have to choose the right species and the right unit. No extra allometric saling required here and I would use the PK-sim inbuild liver wight. However, you are usinge a derived calculated plasme Cl as input to calculate a intrinsic liver clearance back again. In modelling terms this might be not a straight forward approach. However, for a basic check if your hepatocyte CL is in the range of in vivo CL or if you are missing a process, this might be already enough.
The best way (or most pure in terms of IVIVE) would be to get the halflive in hepatocyte and put it in here with the respective assay parameters:
grafik
So you have the ask the lab for the hepatocyte halflive of your compound if you want a more pure IVIVE.
Hope this might help you.

Best Tobias

@Angelefleury

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Angelefleury commented May 24, 2018

Dear colleagues,

I thank you very much for your input! I am extremely grateful for your answers that may be very complicated reading all together, but actually very helpful.

I think my issue is that I have information relative to several items of the metabolism module of PK-Sim but not to a single scaling workflow:
• I have hepatocyte data with/without a strong CYP 3A4 inhibitor, thus I can derive the CYP 3A4 CL from these hepatocyte data assuming that CYP 3A4 is completely inhibited in the experiment (@tobiasK2001 and all: I did not mention this in my first message).

• But unfortunately I cannot enter this value in a microsomal system (as suggested by @krinaj and @msevestre , and commented by @StephanSchaller ) as it was not measured in microsomes. A practical solution would be the one from Stephan, ie. scaling considering how much mg proteins we have in a million hepatocytes (what you mention when writing “volume/cell and mg mic.protein/cell” right?), but to my opinion it is a manipulation that may introduce errors (ie. it is a workaround instead of a straight forward scaling, Tobias is also touching this point). I did this exercise for the sake of understanding (screenshots and explanations below) but it was not conclusive.
If I may ask: we have in PK-Sim microsomal CL in uL/min/mg protein available, why wouldn’t we also have hepatocyte CL in uL/min/Million cells? In that sense I am also expressing interest in the question #101 regarding IVIVE workflow improvement in PK-Sim.

• The Total Hepatic Clearance option would be very handy (liver plasma CL or in vitro t1/2 that I actually have) but as I will later have to predict DDI involving CYP 3A4 metabolism, I have to define which proportion of the CL is driven by CYP 3A4.

Considering this and your various inputs, I understand that I am doing correctly by scaling as follows:
image

taking the scaling factors from Krina, and simply converting uL into L. I then should enter 0.0825 l/min in the “intrinsic clearance – first order” process type, in the “intrinsic clearance” calculation parameters. That way I do not double scale or unscale what PK-Sim does, do I?

Lastly, I tried the workaround of “converting” my microsomal CL into a hepatocyte CL to see what the PK-Sim output would be.
I have 0.38 uL/min/Mio cells in hepatocytes
I consider the table above and scale to a human: 0.0825 l/min to be entered in the intrinsic CL box from the intrinsic Cl process
I get CL spec = 0.0524 1/min:

image

and it results in a plasma CL of 1.32 mL/min/kg (red curve below). Of course I assume no other CL pathways (hepatic or extra hepatic).

Then in a new record I use the workaround:
considering 139 Mio cells/g liver and
considering 40 mg microsomal protein / g liver,
it means 139 Mio cells per 40 mg protein.
So having 0.38 uL/min/Mio cells as above, it means 0.38 * 139 / 40 uL/min/mg protein = 1.3205 uL/min/mg protein
I enter the intrinsic CL in microsomes in the intrinsic CL box
I get CL spec/enzyme = 0.0122 l/umol/min:
image

and it results in a plasma CL of 0.34 mL/min/kg, still considering no other Cl pathways (green curve below). By the way, if I scale manually this CLint = 1.3205 uL/min/mg protein, using Liver blood flow and well stirred model, assuming Blood/plasma 1 as in PK-Sim and fraction unbound assay = 0.97 (calculated by PK-Sim), I get 0.23 mL/min/kg plasma CL, different from the built-in scaling.

You see the result below:
image

so I understand that the workaround is not so easy and we would need to adjust other scaling factors, as I should get the same plasma CL.
I would say:

  • if we have microsomal data we should use the microsomal CL process available.
  • if we have hepatocyte data we have to use the intrinsic CL first order process and enter the CL in l/min as scaled by Krina : Raw CL * Hepatocellularity * Liver weight

Do we have a common understanding?

Best regards

Angèle

@tobiasK2001

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tobiasK2001 commented May 25, 2018

Dear Angèle,

yes I think in principle we can agree on that. Your example shows very nicely that it might not be a good idea to scale from hepatoicyte in vitro data to microsomes.

Just one addidion regarding the scaling: I am not sure vere the liver weight value from krinaj comes from. For consistency reasons I would use the one from PK-sim: Liver volume is here 2.376 l. PK-sim assumes a general organ density of 1 so that would be 2.376 kg. As you assay uses only hepatocytes we should use only the fraction celullar for scaling. 2.3760.667 = 1.584 which is actually almost krinaj value (1561), but just for consistency I would take this one as PK-sim uses this value to normalise your intrinsic CL input to the specific clearance (Clint/(VlivF_cell).

Of course this wourkflow uses significant assumption like:

  • fraction undound in your assay = fraction undound in liver cell
  • substrate concentration in assay is same as in liver cell
  • process is not concentration dependent.

I am not sure if we can hold these and I am afraid these are the persistant trouble makers for IVIVE like e.g. here #125

Best regards, Tobias

@krinaj

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krinaj commented May 26, 2018

Hi Angele and Tobias,

The liver weight that I mentioned earlier just comes from Google. I don't remember exact reference.
I agree with Tobias's approach to calculate liver weight as well.

K

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