siRNA-mediated knockdown of P450 oxidoreductase in rats: a tool to reduce
metabolism by CYPs and increase exposure of high clearance compounds
-Supplementary MaterialRob S. Burke, Inthirai Somasuntharam, Paul Rearden, Duncan Brown, Sujal V. Deshmukh,
Martha A. DiPietro, Jillian DiMuzio, Roy Eisenhandler, Scott E. Fauty, Christopher Gibson,
Marian E. Gindy, Kelly A. Hamilton, Ian Knemeyer, Kenneth A. Koeplinger, Hae Won Kwon,
Traci Q. Lifsted, Karsten Menzel, Mihir Patel, Nicole Pudvah, Deanne Jackson Rudd,
Jessica Seitzer, Walter R. Strapps, Thomayant Prueksaritanont, Charles D. Thompson,
Jerome H. Hochman, Brian A. Carr
PK model development
According to Pang and Rowland, the well-stirred model of hepatic clearance is a good fit in situations
where the drug distribution equilibrium is achieved so rapidly that the unbound drug concentrations in
the emergent venous blood and liver are equal (27). The well-stirred model is characterized by:
𝐸𝑅𝐻 =
𝑓𝑏 ⋅ 𝐶𝐿𝑖𝑛𝑡,𝑢
𝑄𝐻 + 𝑓𝑏 ⋅ 𝐶𝐿𝑖𝑛𝑡,𝑢
(4)
where ERH is the hepatic extraction ratio, fb is the fraction of unbound drug in the blood (i.e. the ratio of
unbound drug concentration in plasma to whole blood drug concentration), CLint,u is the unbound
hepatic intrinsic clearance of drug, and QH is the hepatic blood flow. Instead of a single well-stirred
compartment, the parallel tube model assumes the liver is composed of identical cylindrical tubes and is
a good fit in circumstances where there is a concentration gradient of drug down the length of the tube
(27). In the situation where the unbound plasma concentration of drug (Cu) is much less than the Km of
the CYP-mediated hydroxylation reaction that accounts for loss of parent drug, the equation for the
parallel tube model reduces to:
𝐸𝑅𝐻 = 1 −
−𝑓 ⋅𝐶𝐿
( 𝑏 𝑖𝑛𝑡,𝑢 )
𝑄𝐻
𝑒
(5)
1
In both models, the following definitions hold true:
𝐹𝐻 = 1 − 𝐸𝑅𝐻
(6)
𝐶𝐿𝑏,ℎ = 𝑄𝐻 ⋅ 𝐸𝑅𝐻
(7)
where FH is the fraction of drug that passes through the liver without being metabolized and CLb,h is the
steady-state hepatic blood clearance of drug. Assuming the unbound concentration of drug in blood
cells is equal to the unbound drug concentration in plasma (28), the unbound fraction of drug in blood
(fb) can be determined by:
𝑓𝑏 =
𝑓𝑢,𝑝
𝐶𝑢 𝐶𝑝 ⋅ 𝑓𝑢,𝑝
=
=
(𝐵/𝑃)
𝐶𝑏
𝐶𝑏
(8)
where Cb is the whole blood drug concentration, Cp is the plasma drug concentration, fu,p is the fraction
of drug unbound in plasma defined as fu,p = Cu / Cp, and (B/P) is the blood-to-plasma ratio defined as
(B/P) = Cb / Cp. In a similar fashion, the total blood clearance (CLb) can be determined from in vivo
plasma clearance following an intravenous (i.v.) dose:
𝐶𝐿𝑏 =
𝐶𝐿𝑝
(𝐵/𝑃)
(9)
where CLp is the total plasma drug clearance calculated using the dose and the area under the plasma
drug concentration-time curve (AUC) and is defined as CLp = dose / AUCp,iv. Additionally, when
intrinsic clearance is measured in liver microsomes in vitro, it must be corrected for any effect of
binding to microsomal protein to determine the unbound intrinsic clearance as follows:
𝐶𝐿𝑖𝑛𝑡,𝑢 =
𝐶𝐿𝑖𝑛𝑡,𝑎𝑝𝑝
𝑓𝑢,𝑚𝑖𝑐
(10)
where CLint,app is the apparent intrinsic clearance determined in vitro by eq. 3 and fu,mic is the fraction of
drug unbound in the microsomal incubations (29). The above equations focused on clearance are
predominantly useful following i.v. administration when the total drug dose can be guaranteed to have
2
access to the systemic blood compartment. However, when drugs are dosed orally (p.o.) it becomes
important to understand the bioavailability (F) of the dose (i.e. the fraction of the dose that actually
enters systemic blood circulation). Bioavailability can be experimentally determined as the ratio of the
dose-normalized oral plasma AUC to the dose-normalized i.v. plasma AUC as follows:
𝐹=
𝐴𝑈𝐶𝑝,𝑜𝑟𝑎𝑙
𝐷𝑜𝑠𝑒𝑜𝑟𝑎𝑙
𝐴𝑈𝐶𝑝,𝑖𝑣
𝐷𝑜𝑠𝑒𝑖𝑣
(11)
When assuming that the liver and the gut are the only sites of drug metabolism, the bioavailability
following an oral dose can be defined as:
𝐹 = 𝐹𝐴 ⋅ 𝐹𝐺 ⋅ 𝐹𝐻
(12)
where FA is the fraction of drug absorbed into the gut following an oral dose and FG is the fraction of the
dose that passes through the gut wall without being metabolized (similar to FH defined above). The total
area under the blood drug concentration-time curve following a single oral dose (AUCb,oral) can then be
calculated from the oral plasma AUC as follows:
𝐴𝑈𝐶𝑏,𝑜𝑟𝑎𝑙 = (𝐵/𝑃) ⋅ 𝐴𝑈𝐶𝑝,𝑜𝑟𝑎𝑙
(13)
where AUCp,oral is the experimentally determined oral plasma AUC used in eq. 11. The oral blood AUC
is also defined by:
𝐴𝑈𝐶𝑏,𝑜𝑟𝑎𝑙 =
𝐹 ⋅ 𝑑𝑜𝑠𝑒
𝐶𝐿𝑏
(14)
3
Changes to midazolam PK following POR knockdown
Using the parallel tube model to describe i.v. blood clearance, upon substituting eq. 5 into eq. 7 we find:
−𝑓𝑏 ⋅𝐶𝐿𝑖𝑛𝑡,𝑢
)
𝑄𝐻
(
𝐶𝐿𝑏,ℎ = 𝑄𝐻 ⋅ (1 − 𝑒
)
(15)
Furthermore, for PBS-treated rats the intrinsic clearance of midazolam is unchanged and equal to CLint,u,
while for POR-treated rats there is a 9-fold reduction in the intrinsic clearance of midazolam such that
CLint,u,POR = CLint,u / 9. Determining the ratio of blood clearances, we find:
−𝑓𝑏 ⋅𝐶𝐿𝑖𝑛𝑡,𝑢
)
9⋅𝑄𝐻
)
−𝑓𝑏 ⋅𝐶𝐿𝑖𝑛𝑡,𝑢
)
𝑄𝐻
)
(
𝐶𝐿𝑏,ℎ,𝑃𝑂𝑅
=
𝐶𝐿𝑏,ℎ,𝑃𝐵𝑆
(1 − 𝑒
(
(1 − 𝑒
(16)
Since the limit of the natural exponential function is zero as CLint,u goes to infinity, it is clear from eq. 15
that CLb,h approaches QH at high values of CLint,u for both PBS-treated rats as well as POR-treated rats.
From eq. 16, it is clear that the ratio of the blood clearances then approaches 1 at high values of CLint,u.
In contrast, using the parallel tube model to describe the bioavailability of a p.o. dose, upon substituting
eq. 5 into eq. 6 and then eq. 6 into eq. 12, as well as using FA = FG = 0.95 as described below, we find:
𝐹 = 0.9 ⋅ 𝐹𝐻 = 0.9 ⋅
−𝑓𝑏 ⋅𝐶𝐿𝑖𝑛𝑡,𝑢
(
)
𝑄𝐻
𝑒
(17)
Using the same treatment of the 9-fold reduction in midazolam CLint,u,POR described above as well as the
mathematical definition that ex / ey = e(x - y), we find:
−𝑓 ⋅𝐶𝐿
𝐹𝑃𝑂𝑅
( 𝑏 𝑖𝑛𝑡,𝑢
9⋅𝑄𝐻
=𝑒
𝐹𝑃𝐵𝑆
−
−𝑓𝑏 ⋅𝐶𝐿𝑖𝑛𝑡,𝑢
)
𝑄𝐻
=
8⋅𝑓 ⋅𝐶𝐿
( 𝑏 𝑖𝑛𝑡,𝑢 )
9⋅𝑄𝐻
𝑒
(18)
Thus, the ratio of the bioavailability in rats with POR knockdown to the bioavailability in PBS-treated
rats increases exponentially with increasing CLint,u.
4
Table IV. Experimentally measured and calculated parameters for midazolam used as model inputs.
PBS
Control
anti-POR
siRNA-04
Parameter
Symbol
Units
fraction of unbound drug in plasma
fu,p
0.04 ± 0.002
-
blood to plasma ratio
(B/P)
0.81 ± 0.02
-
fraction of unbound drug in blood (eq. 8)
fb
0.05
-
apparent intrinsic clearance (unscaled)
CLint,app
1973 ± 211
225 ± 13
μL/min/mg protein
apparent intrinsic clearance (scaled)
CLint,app
3551 ± 380
406 ± 23
mL/min/kg
fraction of unbound drug in microsomes
fu,mic
unbound intrisic clearance (eq. 10)
CLint,u
hepatic blood flow rate
QH
70
mL/min/kg
midazolam molecular weight
MW
326
g/mol
i.v. dose
Doseiv
0.5
mg/kg
p.o. dose
Doseoral
10
mg/kg
p.o. dose
Doseoral
30675
nmol/kg
plasma AUC following i.v. dose
AUCp,iv
0.49 ± 0.01
0.68 ± 0.07
µM·h
plasma AUC following p.o. dose
AUCp,oral
0.19 ± 0.05
3.85 ± 1.12
µM·h
total plasma drug clearance
CLp
52.0 ± 1.2
38.5 ± 3.8
mL/min/kg
total blood drug clearance (eq. 9)
CLb
64.1
47.4
mL/min/kg
total blood drug clearance
CLb
3844
2846
mL/hr/kg
fraction absorbed into gut after p.o. dose
FA
0.95
-
fraction of drug unmetabolized through gut
FG
0.95
-
bioavailability (eq. 11)
F
0.02
0.28
-
blood AUC following p.o. dose (eq. 13)
AUCb,oral
0.15
3.13
µM·h
blood AUC following p.o. dose (eq. 14)
AUCb,oral
0.15
3.05
µM·h
0.95
-
3738
427
mL/min/kg
Parameter values are reported as mean ± S.E.M. for those that were experimentally measured. The
values with no reported error were either estimated or calculated using the mean value of the
experimentally determined parameters. A “-” in the units column denotes a dimensionless parameter.
Table V. Parameters determined for midazolam using two models of liver clearance.
Well-Stirred Model
Parallel Tube Model
Parameter
Symbol
PBS
Control
anti-POR
siRNA-04
PBS
Control
anti-POR
siRNA-04
Units
hepatic extraction ratio (eq. 4 or 5)
ERH
0.73
0.23
0.93
0.26
-
hepatic fraction of drug unmetabolized (eq. 6)
FH
0.27
0.77
0.07
0.74
-
hepatic blood clearance (eq. 7)
CLb,h
51.0
16.4
65.2
18.5
mL/min/kg
bioavailability (eq. 12)
F
0.25
0.69
0.06
0.66
-
blood AUC following p.o. dose (eq. 14)
AUCb,oral
2.0
7.4
0.5
7.2
µM·h
The parameter values were calculated using the mean value of the experimentally determined
parameters. A “-” in the units column denotes a dimensionless parameter.
5
PK model discussion
In order to determine the unbound intrinsic clearance (CLint,u) using eq. 10, the fraction of unbound drug
in the microsomal incubations (fu,mic) was determined. For midazolam, it was found that fu,mic = 0.89 ±
0.03 in a microsomal incubation with 0.5 mg/mL of microsomal protein. Similar values can be found
for the fu,mic of midazolam in the literature and it is also known that fu,mic increases as the microsomal
protein concentration decreases due to the presence of fewer protein binding sites (29, 30). At the
microsomal protein concentration of 0.1 mg/mL used in the in vitro incubations in this work the fu,mic
should be close to unity, so an estimated value of 0.95 was used to account for the possibility of a small
amount of protein binding (Table IV).
Since the intrinsic clearance represents the maximum rate of midazolam metabolism in liver
independent of other factors (e.g. hepatic blood flow), any decrease in the intrinsic clearance would lead
to lower rates of hepatic metabolism and manifest as a lower hepatic extraction ratio (ERH). The
fraction of drug that passes through the liver unmetabolized (FH) is defined by eq. 6 such that any
decrease in ERH directly leads to an increase in FH. The total bioavailability (F) defined by eq. 12 is
directly proportional to FH, so any increase in FH directly increases F as well. It was assumed here that
POR knockdown would have no impact on the amount of midazolam absorbed through the gut
following an oral dose and that metabolism of midazolam only occurs in liver and gut tissue. It is clear
from the literature that there is significant intestinal metabolism of midazolam following an oral dose in
humans (31-33); however, it is known for rats that intestinal metabolism is minimal and that FA and FG
for an oral dose of midazolam are close to unity (34-36). In this work, there was no POR knockdown
detected in the mucosal layers of the small intestine (Figure 7), which means that the CYP enzymes in
the enterocytes should retain their full levels of activity and leads to the assumption that FG is not
affected by anti-POR siRNA treatment. Furthermore, when midazolam was incubated with intestinal
6
microsomes, no metabolism was observed even when high microsomal protein concentrations were
used. Taken together, the results presented here are consistent with minimal contributions to overall
bioavailability from FA and FG such that changes in F will be dominated by variations in FH. Thus, both
FA and FG were estimated to be 0.95 to account for nearly complete absorbance and only slight
metabolism in the gut (Table IV).
To understand how the reduced midazolam intrinsic clearance measured in vitro following POR
knockdown would impact PK parameters in vivo, midazolam was dosed i.v. and p.o. two weeks after
administration of LNPs or PBS. Various PK parameters were measured and other PK parameters were
calculated using pharmacokinetic modeling (Tables IV and V). Two models (the well-stirred model and
the parallel tube model) were used to describe the function of the liver with respect to drug metabolism
and clearance (27). In order to use eq. 5 to describe the parallel tube model, the conditions that Cu « Km
and that fb remains constant must be met (27). Midazolam is metabolized by CYP enzymes (primarily
of the CYP3A family) into both 1-hydroxymidazolam and 4-hydroxymidazolam, with the 1hydroxymidazolam formation reaction having a Km ~ 4 μM and the 4-hydroxymidazolam formation
reaction having a Km approximately 10-fold higher in several species including rats (37-40). The
fraction unbound in plasma (fu,p) was measured to be 0.04 for midazolam (Table IV) and is consistent
with other reported values (36, 41). Since Cu = Cp·fu,p and the maximum Cp (from Figure 6) is
approximately 1 μM, the maximum value of Cu is ~0.04 μM. Thus, even using the lowest Km of 4 μM
the maximum value of Cu is 100-fold lower than the Km, which satisfies the Cu « Km constraint. This
constraint also provides that fb can be assumed to remain constant over the length of the tube by
preventing a high enough drug concentration to saturate protein binding sites, which means both
conditions are satisfied and eq. 5 is a reasonable representation of the parallel tube model.
7
As for liver blood flow rate (QH), a value of 70 mL/min/kg was used in the models (Table IV).
This value of QH has been used in other reports (41), but it is slightly lower than the 84 mL/min/kg value
that is typically used for QH in rats. During analysis of the models, it was determined that the lower
value of QH seemed to fit the data more accurately. It is interesting to note that Uhing et al. have shown
that anesthesia with ketamine and xylazine can slow down liver blood flow for several days postsurgery, which can have profound effects on the hepatic clearance of drugs (35). For the animals that
were pretreated with LNPs or PBS prior to dosing of midazolam (Figure 6 and Tables III & IV), jugular
vein cannula surgery was performed in-house using ketamine and xylazine less than 24 h before dosing
midazolam. In contrast, the animals that were dosed with midazolam in the dose-response experiment
(Figure 5 and Table II) were pre-cannulated by the vendor approximately one week before dosing with
midazolam.
The plasma clearance of midazolam in the pre-cannulated animals averaged ~60
mL/min/kg (Table II) while the plasma clearance of midazolam in the in-house cannulated animals
(pretreated with PBS or scramble LNP) averaged ~50 mL/min/kg (Table III), so it could be possible that
the anesthesia slowed down the liver blood flow and reduced hepatic clearance of midazolam. Since
data from the in-house cannulated animals pretreated with LNP or PBS were also used in the PK
modeling (Table IV & V), the use of 70 mL/min/kg for QH was deemed appropriate. Even if a higher
value of QH is used in the analysis, the absolute values of calculated parameters may change, but the
trends and overall conclusions remain the same.
The key findings from the in vivo studies were that POR knockdown leads to a 25% decrease in
plasma clearance that drives a less than 2-fold increase in the plasma AUC following i.v. administration
of midazolam, while POR knockdown leads to a 14-fold increase in bioavailability that drives a 20-fold
increase in plasma AUC following p.o. administration of midazolam. The large difference in response
to POR knockdown depending on the route of administration for midazolam was explored using the two
8
PK models described above. In order to calculate the total blood clearance (CLb) from the total plasma
clearance (CLp) using eq. 9, the blood-to-plasma ratio (B/P) must be determined.
The (B/P) for
midazolam was measured to be 0.81, which is consistent with other reported values (42). The CLb
values (Table IV) were then compared to the values for hepatic blood clearance (CLb,h) calculated using
the two PK models (Table V). The parallel tube model produced CLb,h values that more closely matched
the CLb values than those derived from the well-stirred model, which is not surprising because
midazolam is a high extraction ratio drug (ERH > 0.7) and the parallel tube model generally predicts
behavior more accurately for high ERH drugs. In this work, it was assumed that the vast majority of the
clearance of midazolam is caused by hepatic metabolism, so CLb,h should be nearly equal to CLb. The
fact that both models generated values of CLb,h that were significantly lower than CLb for the animals
with POR knockdown highlights a deficiency in the models. The bioavailability for midazolam in
untreated and PBS-treated animals was determined to be F = 0.02, which is consistent with the literature
(41). POR knockdown led to a significant increase in midazolam bioavailability, and both models
predicted that F would increase upon POR knockdown.
However, the absolute values of the
bioavailability and the increase in bioavailability following POR knockdown compared to PBS
treatment were not accurately predicted by the models. Similarly, the models predicted increased
AUCb,oral following POR knockdown, but again the absolute values and the magnitude of the predicted
changes were slightly different from experimental measurement.
There are a few explanations for the discrepancies between the models and the observations,
including the assumptions regarding QH mentioned above as well as the possibility that the CLint,u
measured in vitro underestimates the actual CLint,u of the enzymes in vivo (the models assume that these
values are equivalent). For both models, the CLb,h asymptotically approaches liver blood flow with
increasing CLint,u, which means that large changes in CLint,u only lead to small changes in CLb for i.v.
9
doses of high clearance compounds (parallel tube model shown in Figure 8). Thus, if the CLint,u for the
hepatic CYP enzymes in vivo were actually higher than those measured in vitro, the same fold reduction
in CLint,u would result in a smaller impact on CLb. Since the CLint,u plays a dominant role in determining
FH, any underestimation of CLint,u would also lead to incorrect predictions of F and AUCb,oral. Despite
the deficiencies of the models, the overall trends from the data are predicted by the parallel tube model:
POR knockdown reduces CYP-mediated metabolism and leads to a reduction in ERH, which leads to an
increase in FH. The increase in FH drives only small reductions in blood clearance following an i.v. dose
of midazolam, but generates large increases in oral bioavailability and blood AUC following a p.o. dose
of midazolam.
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