Visual Overview
Abstract
Pharmacokinetic variability in drug plasma exposure between different studies within the same species is not unexpected due to a variety of factors (such as differences in formulation, active pharmaceutical ingredient salt form and solid-state, genetic strain, sex, environmental, disease status, bioanalysis methods, circadian rhythms, etc.) although variability from within the same research group typically does not occur to a great degree because these variables are commonly controlled. Surprisingly, a pharmacology proof of concept study with a previously validated tool compound from the literature failed to show expected response in murine glucose-6-phosphate isomerase-induced arthritis model which was tied to compound plasma exposure unexpectedly 10-fold lower than exposure observed from early pharmacokinetic study confirming adequate exposure prior to proof of concept. A systematic series of studies were conducted to investigate causes for exposure difference between pharmacology and pharmacokinetic studies identifying the presence or absence of soy protein in animal chow as the causative variable. Cyp3a11 expression in intestine and liver was determined to increase in a time dependent manner in mice switched to diets containing soybean meal compared with mice on diets without soybean meal. The repeated pharmacology experiments using the soybean meal free diet achieved plasma exposures that were maintained above the EC50 and showed efficacy and proof of concept for the target. This effect was further confirmed with marker CYP3A4 substrates in follow on mouse studies. The role of soy protein containing diets on CYP expression necessitates the inclusion of controlling rodent diet as a variable for preventing possible exposure differences between studies.
SIGNIFICANCE STATEMENT The presence of soybean meal protein in murine diet increased clearance and decreased oral exposure for select cytochrome 3A4 substrates. Related effects were also observed on select liver enzyme expression.
Introduction
Soybean meal is often a main component in the chow of laboratory animals, providing a source of complete protein (Zhou et al., 2019). Soy in the diet and orally administered soy extracts have been shown to influence cytochrome P450s (CYPs), UDP-glucuronosyltransferases, drug transporters and transcription factors in multiple species (Bogacz et al., 2014; Křížková et al., 2009; Ronis 2016). Soy products contain isoflavones (e.g., genistein and daidzein) which have been shown to modulate CYP enzymes involved in the metabolism of xenobiotics (Zhou et al., 2019; Marahatta et al., 2014). The laboratory rodent chow diet contains plant-derived ingredients, phytochemicals and supplements, which may influence P450 expression (Zhang et al., 2013).
Regulation of cytochrome P450 gene expression depends upon interactions between xenobiotics and receptors. Likewise, metabolism of xenobiotics is primarily mediated by CYPs and can have a major impact on the bioavailability of a drug affecting its therapeutic efficacy for those drugs that are metabolized by CYPs. When separate groups of male CD1 mice were fed a diet with or without soy protein for 7 days and then dosed intravenously with a test compound at 1 mg/kg, the soy diet mice showed ∼2.4- to 2.9-fold increases in systemic clearance.
The impetus of the work presented here was the lack of efficacy in mouse pharmacodynamic studies for compounds being investigated as tumor necrosis factor (TNFα) modulators. Upon further investigation, it was shown that exposure in the pharmacodynamic (PD) studies was not reproduced from earlier pharmacokinetic (PK) studies. As a result, differences in formulation preparation, lot particle size, mouse strain, mouse vendor, collection tube, anticoagulant and bioanalysis artifacts were carefully investigated and eliminated as the cause of discrepancy between observed PK and efficacy exposures of test compounds at two sites. Diet composition, specifically the presence/absence of soy protein, was identified as the probable cause of exposure differences. The PK and PD studies had been conducted at different laboratory locations and it was identified that the mouse maintenance diet was different between the two sites. Upon further investigation of the four internal global sites conducting rodent PK or efficacy studies, two were using diets containing soy and two were using non-soy containing diets.
Further PK studies were conducted using probe compounds that were predominantly CYP3A4 substrates, similar to the series of compounds being investigated internally. For most substrates, oral administration amplified the differences compared with IV dosing, revealing up to a fourfold decrease in area under the curve (AUC) exposure when comparing mice on soy-based diets to those without soy.
Multiple studies have examined the effects of dietary soy protein and its associated isoflavones on CYP expression and activity as well as xenobiotic metabolism, including comprehensive reviews of the literature (Ronis 2016; Zhou et al., 2019). This study further characterizes the effect of soy protein in the murine diet on the exposures of CYP3A4 substrate tool compounds, itraconazole and gefitinib, and the time-course of the CYP changes in both mRNA and protein levels in mouse liver and duodenum.
Materials and Methods
All animal studies were reviewed and approved by AbbVie's Institutional Animal Care and Use Committee or Oversight Body (in accordance with nation regulations). Animal studies were conducted in an American Association for Accreditation of Laboratory Animal Care accredited program where veterinary care and oversight was provided to ensure appropriate animal care.
Materials
Compound 1 [2-(5-(3-(2,5-Dichlorobenzyl)-2-methylimidazo[1,2-A]pyridin-6-yl)pyrimidin-2-yl)propan-2-ol] (lot 2193407) was provided by Process Chemistry, AbbVie (North Chicago, IL), structure provided in Fig. 1.
Plasma concentration versus time profiles of a TNFα inhibitor compound (structure inset) following 17 oral doses in the PD study versus a single dose in the PK study. PD and PK study dosed in 10:90, v/v, nanosuspension in 2% HPC-SL, 0.2% SDS in water:D5W. Animals in the PD study were dosed using a fixed body weight, while in the PK study, the animals were dosed based on body weight. PD study conducted in DBA/1J male mouse, PK study conducted in CD-1 male mouse (n = 3). Serial blood samples were collected to 24 hours in the PD study using heparin anticoagulant. Serial blood samples were collected to 48 hours in the PK study using EDTA anticoagulant. Studies were conducted at different sites utilizing different mouse chows, but all plasma samples were analyzed at a single site and laboratory.
Quetiapine (lot 2154716, Sigma Chemical), Gefitinib (lot 1908238, AbbVie), Itraconazole (lots 107518 (Janssen Pharmaceuticals), 1984815 (Sigma), 097K1156V (Sigma)) Tadalafil (lot 1066832, AbbVie), were purchased as indicated or provided by Process Chemistry, AbbVie.
Animals and Diets in PK Studies
Male CD-1 mice, weighing 20–25 g (est. 31–44 days old), were obtained from Charles Rivers Laboratories (Wilmington, MA) and were socially housed and allowed free access to food and water. PK studies were conducted in groups of mice; each group contained three animals. All groups were dosed with a solution formulation. Internal TNFα inhibitors were formulated in 1:5:10:74 (v/v) DMSO: Tween 80:PEG-400 (polyethylene glycol 400):D5W (dextrose in water). Comparator CYP3A4 substrate compounds were formulated in 5:5:20:70 (v/v) DMSO:Tween 80:PEG-400:D5W. Dosed animals were switched from the vendor diet to the study diet at least 1 week prior to dosing. Groups of three mice received either an intravenous dose via the penile vein, administered under isoflurane anesthetic or an oral dose, administered by gavage. Sequential blood samples were obtained from a tail vein of each animal for 24 hours after dosing. Plasma was separated by centrifugation and stored frozen until analysis.
Diets used included Teklad 2014 Global 14% Protein Rodent Maintenance Diet (Envigo, Madison, WI), 5010 Laboratory Autoclavable Rodent Diet, 5L79LabDiet, Prolab RMH3000 (LabDiet, St Louis, MO), and Ssniff Rodent Maintenance 2014 (ssniff Spezialdiäten GmbH, Soest, Germany). Diets are described in Table 1 and Table 2.
Standard mouse chows evaluated
Protein normalized chows evaluated
Studies with select CYP3A4 marker compounds were originally dosed using a cassette method with n = 4 compounds per groups of 3 mice per route of administration. Later studies were conducted with individually dosed compounds per group of 3 mice for PK determination.
Animals and Diet in PD Studies
Male DBA1/J mice (Jackson Laboratories, Bar Harbor, ME) were immunized intradermally (i.d.) at the base of the tail with 100 µl of 1:1 (v/v) emulsion containing 300 µg of glucose-6-phosphate isomerase and 200 µg of heat-inactivated Mycobacterium tuberculosis H37Ra (Complete Freund’s Adjuvant), Difco, Lawrence, KS). The mice were dosed orally once daily beginning prior to glucose-6-phosphate isomerase immunization with vehicle and Compound 1 at 10, 30, or 100 mg/kg from days 0–17. Compound 1 was prepared as a 10X nanosuspension stock which was diluted in D5W for dosing daily, with a dose volume of 200 µl per dose. Seven days after immunization, mice were monitored for arthritis. Rear paws were evaluated for paw-edema using Dyer spring calipers on days 7, 10, 13, 15, and 17. Mice began to show signs of paw swelling between day 7 and 10. At the termination of the experiment, a full 24-hour exposure AUC was performed on the final day of dosing for Compound 1.
Mice were DBA1/J from Jackson laboratories, 10 weeks old when the study began, with an average body weight of around 21–22g. Mice were maintained on Diet B for 13 days prior to the study start and throughout the study period.
Plasma Test Article Concentration Analysis
Plasma was spiked with internal standard and then compounds were selectively removed using protein precipitation with acetonitrile at neutral pH. The test article and the internal standard were separated from each other and co-extracted contaminants on either a 50 × 3 mm or 30 × 2.1 mm Fortis Pace C18 5 µm column (Fortis, London, UK) with a mobile phase consisting of acetonitrile:0.1% formic acid at a flow rate of 0.8 ml/min. Analysis was performed on a Sciex API5500 (PE Sciex Applied Biosystems, Foster City, CA) with either a turbo-ionspray or heated nebulizer interface using multiple reaction mode detection. Test article and internal standard peak areas were determined using Analyst software. The concentration of each sample was calculated by least squares linear regression analysis of the peak area ratio (parent/internal standard) of the spiked plasma standards versus concentration.
Calculation of PK Parameters and Statistical Analysis
The plasma concentration data were submitted to multi-exponential curve fitting using WinNonlin (version 5.2, Certara USA Inc., St. Louis, MO). Peak plasma concentration (Cmax) was read directly from the concentration data for each animal. The area under the plasma concentration-time curve from 0 to t hours (time of the last measurable plasma concentration) after dosing (AUC0-t) was calculated using the linear trapezoidal rule for the plasma concentration-time profiles. The residual area extrapolated to infinity, determined as the final measured plasma concentration (Ct) divided by the terminal elimination rate constant (β), was added to AUC0-t to produce the total area under the curve (AUC0-∞). The apparent total plasma clearance was calculated by dividing the administered dose by the AUC0-∞. Data are expressed as mean ± S.D.
Statistical analysis of PK data were performed using propagation of error methods. The T-test (2-sided, unpaired) was used to compare AUC and Cmax values following oral doses or CL values following intravenous doses and the P value determined. PK parameters with a P value less than 0.05 were deemed significant.
In-Life Diet Study
Male CD-1 mice, weighing 20–25 g (est. 31-44 days old), were obtained from Charles Rivers Laboratories (Wilmington, MA) and were socially housed and allowed free access to food (vendor diet, LabDiet 5L79, soy containing) and water. On the day of arrival 25 mice were switched to Diet A (2014 Teklad Global Diet, non-soy containing) and 25 mice were switched to Diet B (LabDiet 5010 soy containing). Five mice on each in-house diet were sacrificed at 2, 5, 7, 14 and 30 days post-switch. Five mice were maintained on vendor diet (soy protein containing) for 2 days post arrival, then sacrificed to obtain baseline values. Samples (liver, duodenum) were analyzed as described below.
Gene Expression Analysis by Real-Time Polymerase Chain Reaction (PCR)
Total RNA was isolated from flash-frozen (liquid nitrogen) mouse liver using the Qiazol (Qiagen, Valencia, CA) method and from mouse duodenum sections, flushed with saline, and harvested in RNAlater (Ambion, Austin, TX) using the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA samples were evaluated for quality using the Agilent 2100 Bioanalyzer platform (Agilent, Santa Clara, CA). Total RNA samples were analyzed by RT-PCR using the iTaq Universal Probes One-Step Kit (Bio-Rad, Hercules, CA) and the Taqman Gene Expression Assays (Applied Biosystems, Carlsbad, CA) for Cyp3a11 (Mm00731567_m1) and Cyp2b10 (Mm01972453_s1) on the ViiA 7 Real-Time PCR system (Applied Biosystems) with the following parameters: 10 minutes at 50°C (RT), 3 minutes at 95°C (polymerase activation), 40 cycles of 15 seconds at 95°C (denaturation), then 1 minute annealing/extending at 60°C annealing/extending. The (ΔΔ Ct) method was used to calculate the fold change of Cyp3a11 or Cyp2b10 mRNA levels normalized to 18S ribosomal RNA (Hs99999901_s1) in tissues from mice fed Diet B compared with mice fed Diet A.
Protein Analysis by Western Blot
Expression of Cyp3a11 protein in mouse livers was evaluated with Western Blot (n = 5). Mouse liver tissue homogenate was prepared according to the manufacturer’s instructions using Tissue Protein Extraction Reagent (ThermoFisher, Waltham, MA) supplemented with Protease Inhibitor Cocktail (Roche, Indianapolis, IN). Total protein concentration was determined using Bradford Assay Reagent (Bio-Rad, Hercules, CA). Total liver protein (25 µg) was run on NuPAGE Novex 4–12% Bis-Tris Protein Gel, and then transferred to PVDF membrane using the iBlot transfer system (Invitrogen, Waltham, MA). The blot was blocked at room temperature for 1 hour (TBS, 0.1% Tween-20 with 5% nonfat dry milk). The blot was incubated at 4°C overnight with primary antibody (1:1200) against Cyp3a11 protein (Mouse Anti-Rat Cytochrome P450 Monoclonal (cat#: MAB10041, Millipore, Billerica, MA). Cyclophilin antibody (Abcam, Cambridge, MA) was used as a loading control for normalization (1:10,000). Fluorescently labeled secondary antibodies (anti-mouse and anti-rabbit, 1:10,000 dilution) were incubated with the blot for 45 minutes at room temperature. The blot was visualized on an Odyssey Imaging system (LI-COR Biosciences, Lincoln, NE), and densitometric analysis of proteins was performed using Image Studio Software (LI-COR Biosciences). Mouse duodenum and jejunum homogenates were also evaluated via western blotting, but no appreciable induction of Cyp3a11 protein was observed.
Statistical Analysis for Expression Data
RNA and densitometry data were analyzed for statistical significance using one-way ANOVA (ordinary) and the T-test (2-tailed, unpaired) in GraphPad Prism (Version 9.1.0).
Results
PK and PD Studies Exposure Differences
PK and PD studies were conducted with a TNFα inhibitor compound (Bentley et al., 2014) at two geographically distinct sites that maintained their mouse colonies on Diet A (PK) or Diet B (PD), respectively. As shown in Fig. 1, the PK study exposure was above the EC50 for approximately 18 hours following a single dose of 100 mg/kg. The PD study dose on Day 17 did not achieve the EC50 level. The PK data shown in Table 3 reveal a greater than 10-fold difference in the exposure after oral doses at two different sites. Likewise, the arthritis model results (not shown) from this PD study indicated a lack of efficacy following 17 daily doses of 100 mg/kg Compound 1. In vitro data suggests Compound 1 is a substrate of human CYP3A4 (data not shown).
Mean pharmacokinetic parameters of Compound 1 following 100 mg/kg oral doses with male mice fed a soy free or soy containing diet for at least 7 days prior to dosing
Effect of Mouse Chow on PK of TNFα Inhibitors
Since multiple factors differed between the PK and PD studies conducted at the two different sites, the pharmacokinetics of Compound 1 were evaluated at a single site using 1 mg/kg doses administered by both intravenous and oral routes. Male CD-1 mice were maintained on either Diet A or Diet B for 7 days prior to dosing. As shown in Table 4, systemic clearance was 2.9-fold higher in mice maintained on a soy containing diet (Diet B) compared with mice maintained on soy-free diet (Diet A). Similarly, plasma AUC of Compound 1 following an oral dose was reduced approximately 5-fold in mice maintained on soy containing Diet B (Table 4, Fig. 2).
Mean pharmacokinetic parameters of Compound 1 following 1 mg/kg intravenous and oral doses with male CD-1 mice fed a soy free or soy containing diet for 7 days prior to dosing
Plasma concentration versus time profiles of a TNFα inhibitor compound following 1 mg/kg intravenous and oral dosing with male mice fed a soy free or soy containing diet (Diet A or Diet B, respectively) for 7 days prior to dosing. Compound 1 dosed as a solution in DMSO:Tween 80:PEG-400:D5W (1:5:20:74, v/v) at 10 ml/kg (Mean ± S.D., n = 3).
Cassette Study with CYP3A4 Marker Substrates
To confirm the initial findings of the effect of soy diet on PK exposure for internal compounds predominantly metabolized by CYP3A4, four additional CYP3A4 substrates were dosed in a cassette experiment to male mice. Doses of 1 mg/kg of itraconazole, tadalafil, gefitinib and quetiapine were administered by intravenous or oral route to groups of three mice in a cassette format. Mice had been on the Diet A or Diet B regimen for at least 7 days prior to dosing. All compounds showed an increase in IV clearance when mice had been fed Diet B compared with Diet A (1.3- to 2.5-fold increase). Following a single oral dose, both Cmax and AUC were decreased for animals fed Diet B compared with Diet A, although the decrease was not significant in the case of quetiapine. The Diet B AUC values were 3.2- to 4.6-fold lower than those found with Diet A following the oral dose (Table 5, Fig. 3).
Pharmacokinetic parameters of test articles obtained from mice fed soy free (A) or soy containing chow (B)
Compounds were dosed as a cassette to n=3 male mice.
Plasma concentration-time profiles of itraconazole, tadalafil, gefitinib, and quetiapine following intravenous and oral dosing in mice fed soy free (Diet A) or soy containing chow (Diet B). Dose was 1 mg/kg of each compound formulated in DMSO:Tween 80:PEG-400:D5W (5:5:20:70, v/v) at 10 ml/kg in a cassette format (n = 3 males per route).
Discrete Study with Itraconazole and Compound 1
Itraconazole was chosen for further evaluation using mice fed diets used at the four global PK/PD sites along with Compound 1. Mice were maintained on one of the four global diets for at least a week prior to dosing groups of three male mice with 1 mg/kg itraconazole or Compound 1 by both the intravenous and oral routes. PK parameters were similar for animals on Diets A and C, both diets without soy. Intravenous clearance was increased (1.5- to 3.0-fold) for mice maintained on Diets B and D, both diets containing soy. Likewise, oral exposure was reduced for animals on the soy-containing diets, as measured by both Cmax and AUC (Table 6, Figs. 4 and 5).
Itraconazole and Compound 1 pharmacokinetic parameters (average ± S.D.) obtained from mice fed four various vender diets and reconstructed diets
Intravenous and oral PK profiles of itraconazole in mice fed Diet A, Diet B, Diet C, or Diet D (Mean ± S.D., n = 3). Dose was 1 mg/kg for each diet and route evaluated. Itraconazole formulated as solution in DMSO:Tween 80:PEG-400:D5W (5:5:20:70, v/v) and dosed at 10 ml/kg. Diets A and C are soy-free; Diets B and D contain soy.
Intravenous and oral PK profiles of Compound 1 in mice fed Diet A, Diet B, Diet C, or Diet D (Mean ± S.D., n = 3). Dose was 1 mg/kg for each diet and route evaluated. Compound 1 formulated as solution in DMSO:Tween 80:PEG-400:D5W (1:5:20:74, v/v) and dosed at 10 ml/kg. Diets A and C are soy-free; Diets B and D contain soy.
Since Diet A was known to contain 20% protein compared with 29% protein in Diet B, Diet A was supplemented (in-house) to equalize the protein content. In one instance, corn gluten was used to increase the protein content, and in the other, soybean meal was the additive. Itraconazole and Compound 1 were again dosed as the test articles, and the results confirmed the lower exposure and increased clearance with animals that were fed diets containing soy (Table 6, Figs. 6 and 7).
Intravenous and oral PK profiles of itraconazole in mice fed protein normalized Diet A (Mean ± S.D., n = 3). Dose was 1 mg/kg for each diet addition and route evaluated. Itraconazole formulated as solution in DMSO:Tween 80:PEG-400:D5W (5:5:20:70, v/v) and dosed at 10 ml/kg.
Intravenous and oral PK profiles of Compound 1 in mice fed protein normalized Diet A (Mean ± S.D., n = 3). Dose was 1 mg/kg for each diet addition and route evaluated. Compound 1 formulated as solution in DMSO:Tween 80:PEG-400:D5W (1:5:20:74, v/v) and dosed at 10 mL/kg.
Gene Expression Analysis by Real-Time PCR
Expression levels of Cyp1a1, Cyp2b10 and Cyp3a11, the 3 main inducible cytochrome P450 mouse genes, were assessed by RT-PCR in total RNA isolated from flash-frozen liver of mice fed four different diets. When compared with mice fed Diet A (non-soy-containing), Cyp3a11 expression was clearly increased (2- to threefold) in mice fed the soy-containing diets (Diets B and D). The mice fed Diet C (non-soy-containing)) showed a similar expression of Cyp3a11 to Diet A. Cyp1a1 did not appear to be affected by the diets to a similar degree. Cyp2b10 expression was too variable among animals to draw a conclusion about the influence of diet (Fig. 8).
Levels of Cyp1a1, Cyp2b10, and Cyp3a11 mRNA in livers from mice fed Diet B (soy-containing), Diet C (non-soy-containing), or Diet D (soy-containing) compared with mice fed Diet A (non-soy-containing), n = 3 mice per diet. Data, generated by RT-PCR, are expressed as fold-change (mean ± S.D.) compared with Diet A as calculated by ΔΔCt method. Asterisks indicate that the values were significantly different compared with Diet A, *P < 0.0001 (one-way ANOVA).
Time-Course Evaluation of mRNA Expression in Mouse Liver and Duodenum
The time course of the effect of diet on Cyp3a11 and Cyp2b10 mRNA expression in liver and duodenum was evaluated by sacrificing groups of mice (n = 5) over a period of 2 to 30 days post diet change (Fig. 9). An additional group of 5 mice were maintained on the vendor diet (soy-containing) for 2 days prior to sacrifice and served as the baseline value. Within 2 days of switching from the vendor diet (soy-containing) to Diet A (non-soy-containing), Cyp3a11 expression in mouse liver decreased. This decrease was sustained out to 30 days. Livers from mice switched to Diet B (soy-containing) maintained similar Cyp3a11 expression levels as vendor baseline over the same test period. Although a similar trend was seen with Cyp2b10 expression, data were too variable for conclusions to be drawn. Minimal differences were observed in mRNA expression of Cyp3a11 and Cyp2b10 in duodena, although the trend was similar to liver (Fig. 10).
Time-course of Cyp3a11 and Cyp2b10 mRNA expression in livers of mice fed Diet A or Diet B. Samples were run in technical duplicates. Data, generated by RT-PCR, are expressed as normalized Ct, threshold cycle normalized to 18S housekeeping gene, and are inversely proportional to expression level, mean ± S.D., n = 5 mice per timepoint for Diet A (non-soy) and Diet B (contains soy). Asterisks indicate that the values were significantly different compared with baseline vendor diet (contains soy), *P < 0.0001 (unpaired two-tailed T-test).
Time-course of Cyp3a11 and Cyp2b10 mRNA expression in duodena of mice fed Diet A or Diet B. Samples were run in technical duplicates. Data, generated by RT-PCR, are expressed as normalized Ct, threshold cycle normalized to 18S housekeeping gene, and are inversely proportional to expression level, mean ± S.D., n = 5 mice per timepoint for Diet A (non-soy) and Diet B (contains soy). Asterisks indicate that the values were significantly different compared with baseline vendor diet (contains soy), *P < 0.0001 (unpaired two-tailed T-test).
Liver Weight Not Affected by Diet
Within the 30-day testing period, no difference was observed in relative liver weight (liver weight as percentage of total body weight) of mice fed Diet A or Diet B (Fig. 11).
Time course of relative liver weights (liver weight as percentage of total body weight) of mice fed Diet A and Diet B. Data are expressed as mean ± S.D., n = 5 mice per timepoint for Diet A and Diet B.
Time-Course Evaluation of Protein Expression Levels in Liver—Analysis by Western Blot
In a similar manner to the mRNA experiment, the protein expression levels in mouse livers were examined over the same time period (2 days and 30 days) after diet switch. Within 2 days of switching to non-soy protein containing diet (Diet A), Cyp3a11 protein expression in mouse liver decreased approximately 10-fold. This decrease was observed to be maintained in the 30-day liver samples as well (Fig. 12).
Effects of dietary change on CYP3A11 Cyp3a11 protein expression in mouse livers. Tissue homogenate was prepared from livers of male CD-1 mice (n = 5) that were fed a vendor diet or were switched from the vendor diet to Diet A or Diet B for 2 to 30 days. (A–B) Western Blot analysis of CYP3A11 Cyp3a11 protein expression. Antibodies used are described in Materials and Methods. (A) Expression of CYP3A11 Cyp3a11 protein in mouse liver 2 days or (B) 30 days after diet switch. Each lane contained 25 µg of total liver protein for each sample analyzed. Cyclophilin B was included as a loading control. Densitometry analysis (conducted with Li-Cor Odyssey Imaging Software) of relative CYP3A11 Cyp3a11 protein levels in livers of mice fed vendor diet, Diet A and Diet B. Asterisks indicate that the values were significantly different compared with Vendor, **P < 0.0001, *P < 0.05 (one-way ANOVA).
Repeated PD Study
Male DBA1/J mice were maintained on Diet A for at least 7 days prior to the initiation of the glucose-6-phosphate isomerase study as described earlier. At the termination of the experiment, a full 24-hour exposure AUC was performed on the final day of dosing for Compound 1. Compound 1 plasma concentrations remained above the EC50 level for approximately 14 hours following daily doses of 100 mg/kg. Efficacy was still not achieved until Compound 1 was delivered on a twice a day schedule (Table 7). When comparing the AUC obtained before the diet switch (Table 3) to the AUC after the switch to Diet A, the exposure was 20-fold greater.
Repeat dose efficacy study conducted with Compound 1 in mice maintained on Diet A throughout study period
Discussion
In today’s global drug development paradigm, efficacy and pharmacokinetic studies may be conducted at geographically remote sites, or even at different entities if early discovery work is outsourced. To maintain consistency in data obtained from studies conducted at different sites, care must be taken to ensure as many variables as possible remain tightly controlled. Our results demonstrate that the rodent maintenance diet significantly impacted PK parameters when mice were dosed either intravenously or orally with a series of test compounds that were predominantly CYP3A4 substrates.
The impact of soy in the diet has been recognized in recent years as having an effect on various liver and intestinal components involved in xenobiotic metabolism. In a series of studies in rats, the impact of soy on hepatic CYP3A was investigated from weaning to adulthood (Ronis et al., 1999; Ronis et al., 2004; Ronis et al., 2006). It was shown that effects on CYP3A1 expression appeared to be due to phytochemical components of soy protein isolates other than isoflavones, but that soy protein and the isoflavone daidzein were involved in the induction of CYP3A2 (Ronis et al., 2006).
In a study by Li, the impact of the primary isoflavones in soy (genistein, daidzein, and the daidzein metabolite equol) were studied in murine Cyp3a11 expression. Wild-type mouse hepatocytes showed induction of Cyp3a11 mRNA following genistein and daidzein treatment, but no effect with equol. The same induction effect was observed in vivo when male mice were fed a soy protein-containing diet as compared with a diet that had been stripped of isoflavones by ethanol washes (Li et al., 2009).
Zhang et al. investigated the expression of liver and intestinal CYPs in WT and IE-Cpr-null mice fed a soy-containing rodent chow and a synthetic diet devoid of phytochemicals. That study showed elimination of soy from the rodent diet resulted in a decrease in expression of CYP1A, 2B, 2C, and 3A in the small intestine and CYP2B, 2C, and 3A in livers of both WT and IE-Cpr-null mice. In addition, it was shown that in WT mice, a change to the synthetic diet (non-soy) resulted in a ∼2-fold increase in AUC for orally administered midazolam, a CYP3A4 marker substrate (Zhang et al., 2013). Similar results were shown in a study by van Waterschoot, where replacing a standard laboratory diet with a semisynthetic diet (with reduced phytochemicals) decreased hepatic expression levels of murine Cyp3a11 and Cyp3a25 genes by ∼1.5-to 3-fold (van Waterschoot et al., 2009).
In the present study, male CD1 mice were fed site-specific maintenance rodent chows to investigate differences in PK parameters that had been observed in PD versus PK studies with a series of proprietary compounds. To further elucidate the effect of diet on PK parameters, a collection of CYP3A4 marker substrates were dosed both intravenously and orally in mice fed a series of four standard commercial rodent chows. Two of the chows contained soy and two were non-soy containing.
In a cassette-dosed study of itraconazole, tadalafil, gefitinib, and quetiapine, all showed increased intravenous clearance and decreased oral exposure when animals fed a soy-containing diet were compared with those maintained on a non-soy containing diet.
To further elucidate the impact of diet on PK, itraconazole was chosen as a marker CYP3A4 substrate for further diet studies, along with Compound 1. Similar PK parameters were obtained in mice maintained on diets that were free of soy (Diets A and C) while noticeable differences were observed in mice maintained on diets containing soy (Diets B and D). Following a single intravenous dose, the intravenous clearance of both compounds was increased in animals maintained on soy containing diets by a factor of 1.5- to 3.0-fold. Likewise following a single oral dose, exposure was reduced in mice on the soy containing diet, up to a factor of 3.6-fold for itraconazole and 6.7-fold for Compound 1.
Since the composition of the standard diets slightly differed in the kcal percent provided by protein, fat, and carbohydrates, Diet A (non-soy) was supplemented to standardize the protein content with either the addition of soybean meal or a corn gluten/starch combination. Single intravenous and oral doses of itraconazole and Compound 1 again confirmed the impact that soy has on the exposure. In this study, the mice on the diet supplemented with soybean meal showed a 1.9-fold increase in intravenous clearance and a 2.4-fold decrease in oral exposure for itraconazole and a 2.7-fold increase in intravenous clearance and a 2.8-fold decrease in oral AUC for Compound 1.
To investigate the xenobiotic systems mediating the PK exposure changes, liver weight, mRNA and protein expression were all investigated in the mouse. In an early study with the internal proprietary compounds, the mRNA expression in liver was shown to be similar in mice fed non-soy diets (Diets A and C), but clearly induced for Cyp3a11 in mice fed soy-containing diets. No difference could be ascertained in the mRNA expression for Cyp1a1, and the Cyp2b10 expression was too variable to reach a conclusion about the influence of diet.
Further time course studies were conducted to evaluate the mRNA changes in mouse liver and duodenum tissue. The change in liver expression of Cyp3a11 occurred as soon as 2 days post diet adjustment for mice switched from the vendor diet (containing soy) to a diet free from soy (Diet A). For mice that were switched to Diet B, which also contains soy, the expression of Cyp3a11 in liver was very similar to that found with the vendor diet. A similar trend was observed for Cyp2b10 in mouse liver, but data were too variable for conclusions to be drawn. While the duodenum tissues from mice fed Diet B were consistently higher than those for Diet A in the expression of both Cyp3a11 and Cyp2b10, the data were more variable than that from liver and no conclusions could be drawn. These results show that changes in mRNA expression in the liver by soy containing diets occur rapidly.
To further investigate the changes observed in Cyp3a11, the protein expression levels in mouse liver were also measured. Within 2 days of switching the mice from the vendor diet (which contained soy) to Diet A (non-soy-containing), Cyp3a11 protein expression decreased approximately 10-fold. Similar results were observed in the mouse liver 30 days after the diet switch, and thus the effect appeared to be sustained throughout the study.
The pronounced soy-protein effects observed on CYP expression in mice reported here and elsewhere have not generally been reported to be observed clinically in humans, although some studies using pure soy isoflavones have suggested some modest increase in CYP3A (Ronis 2016). The lack of soy-protein drug interactions in humans compared with rodents may be due to species differences in drug-activated nuclear receptors (e.g., PXR) or greater genetic and environmental variability in humans masking soy-protein drug interactions compared with inbred mouse strains in a laboratory.
While there is extensive literature on the effect of soy in the diet on rat CYPs, this is the first study to examine multiple diets and their effects on PK of CYP3A4 substrates, mRNA, and protein expression in mice. In conclusion, our studies revealed the following points:
PK exposure differences were linked to differences in diets used.
Mice on soybean meal diets had reduced PK exposures for CYP3A4 substrate compounds compared with mice on diets without the soybean meal.
PK exposure differences were seen after both intravenous and oral dosing routes, with larger differences in the orally dosed mice.
Normalizing protein content in the diets by adding soybean meal to Diet A resulted in mice with lower plasma exposures than mice fed Diet A with corn gluten meal added.
Livers from mice fed Diets A, B, C, and D were evaluated, and mice on diets containing soybean meal showed increased expression of Cyp3a11.
While the source of protein in a standard rodent diet may not typically be considered when planning studies, we have shown that when noting variability in pharmacokinetic exposures across studies it is important to determine whether a soy-containing diet is impacting pharmacokinetic efficacy and potentially safety due to the effect on CYP expression.
Acknowledgments
The authors would like to thank Mary Larsen for bioanalytical support, David Cugier for mRNA isolation support, and Darlene Satonin for manuscript preparation assistance.
Data Availability
The authors declare that all the data supporting the findings of this study are either contained within the paper or are available upon request from the corresponding author.
Authorship Contributions
Participated in research design: Wetter, Ciurlionis, Liguori, Goess, Mathieu, Kalvass.
Conducted experiments: Wetter, Ciurlionis, Krause, Mathieu.
Performed data analysis: Wetter, Ciurlionis, Krause, Goess, Mathieu, Kalvass.
Wrote or contributed to the writing of the manuscript: Wetter, Ciurlionis, Krause, Liguori, Goess, Mathieu, Kalvass.
Footnotes
- Received January 5, 2023.
- Accepted April 17, 2023.
AbbVie sponsored and funded the study; contributed to the design; participated in the collection, analysis, and interpretation of data, and in writing, reviewing, and approval of the final publication. This work received no external funding.
All authors were employees of AbbVie at the time the work was conducted and may own AbbVie stock.
Abbreviations
- AUC
- area under the concentration time curve
- Compound 1
- [2-(5-(3-(2,5-Dichlorobenzyl)-2-methylimidazo[1,2-A]pyridin-6-yl)pyrimidin-2-yl)propan-2-ol]
- CYP
- cytochrome P450 isoform
- D5W
- 5% dextrose in water
- PCR
- polymerase chain reaction
- PD
- pharmacodynamic
- PEG-400
- polyethylene glycol 400
- PK
- pharmacokinetic
- PO
- oral
- P450
- cytochrome P450
- QD
- once daily
- Soy
- soybean meal or soy protein containing
- Soy-free
- no soybean meal or protein
- but may contain soybean oil
- TNFα
- tumor necrosis factor
- Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics
