Abstract
Antiretroviral drug (ARV) metabolism is linked largely to hepatic cytochrome P450 activity. One ARV drug class known to be metabolized by intestinal and hepatic CYP3A are the protease inhibitors (PIs). Plasma drug concentrations are boosted by CYP3A inhibitors such as cobisistat and ritonavir (RTV). Studies of such drug-drug interactions are limited since the enzyme pathways are human specific. While immune-deficient mice reconstituted with human cells are an excellent model to study ARVs during human immunodeficiency virus type 1 (HIV-1) infection, they cannot reflect human drug metabolism. Thus, we created a mouse strain with the human pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 genes on a NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac background (hCYP3A-NOG) and used them to evaluate the impact of human CYP3A metabolism on ARV pharmacokinetics. In proof-of-concept studies we used nanoformulated atazanavir (nanoATV) with or without RTV. NOG and hCYP3A-NOG mice were treated weekly with 50 mg/kg nanoATV alone or boosted with nanoformulated ritonavir (nanoATV/r). Plasma was collected weekly and liver was collected at 28 days post-treatment. Plasma and liver atazanavir (ATV) concentrations in nanoATV/r-treated hCYP3A-NOG mice were 2- to 4-fold higher than in replicate NOG mice. RTV enhanced plasma and liver ATV concentrations 3-fold in hCYP3A-NOG mice and 1.7-fold in NOG mice. The results indicate that human CYP3A-mediated drug metabolism is reduced compared with mouse and that RTV differentially affects human gene activity. These differences can affect responses to PIs in humanized mouse models of HIV-1 infection. Importantly, hCYP3A-NOG mice reconstituted with human immune cells can be used for bench-to-bedside translation.
Introduction
Humanized immunodeficient mice are now considered a suitable model for studies of human immunodeficiency virus (HIV) type 1 (HIV-1) disease pathobiology and therapy (McCune et al., 1990; Merry et al., 1997; André et al., 1998; Riska et al., 1999; Limoges et al., 2001; Stoddart et al., 2007; Denton et al., 2008; Donia et al., 2010; Sango et al., 2010; Dash et al., 2012; Nischang et al., 2012; Roy et al., 2012; Speck, 2015). To serve such goals CIEA NOG (Ito et al., 2002) or NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (NOD scid gamma mice; The Jackson Laboratory; Bar Harbor, ME) are reconstituted with human CD34+ hematopoietic stem cells (Ishikawa et al., 2005; Gorantla et al., 2012; Gonzalez et al., 2013; Marsden and Zack, 2017). Work in our own laboratory and those of others have shown the application of this animal model for testing long-acting antiretroviral (ARV) drug nanoformulations developed for both pre-exposure prophylaxis and treatment of existing infections (Dash et al., 2012; Puligujja et al., 2015a; Kirtane et al., 2016; Zhang et al., 2016; Edagwa et al., 2017; Zhou et al., 2018). Prior studies on nanoformulated ritonavir-boosted atazanavir (nanoATV/r) administered weekly demonstrated sustained plasma drug concentrations and suppressed HIV-1 infection with concomitant restoration in CD4+ T-cell numbers (Dash et al., 2012). Although humanized mice serve as valuable tools to assess antiretroviral efficacy, divergences in drug metabolism between humans and rodents makes translation of studies from rodents to humans difficult.
Most ARV interactions occur through inhibition or induction of hepatic drug metabolism. These are linked, in the largest measure, to the cytochrome P450 (P450) monooxygenase system. The enzymes are transcriptionally regulated by ligand-activated transcription factors such as the aryl hydrocarbon receptor, pregnane X receptor (PXR), and constitutive androstane receptor (CAR) (Handschin and Meyer, 2003). Similar to the P450 enzymes, PXR and CAR can bind multiple different ligands and their ligand specificity varies across species (Timsit and Negishi, 2007). Overlapping ligand specificity between PXR and CAR, species specificity in ligand binding, and the opposing effects of these receptors’ activities make the net effect of xenobiotic regulation of major P450s complex (Moore et al., 2000). For HIV-1 pharmacology there are challenges associated with multiple drug combinations. Thus there is a need to characterize, understand, and limit drug-drug interactions in the context of human drug metabolism.
As a clinically relevant example, for the HIV-1 protease inhibitors (PIs) lopinavir, atazanavir (ATV), and darunavir, intestinal absorption and hepatic elimination are largely governed by CYP3A4/5 activity (Shen et al., 1997). PI pharmacokinetic profiles are boosted by ritonavir (RTV) or cobicistat primarily through their inhibition of CYP3A enzymatic activity (Hull and Montaner, 2011). When used in HIV-1 treatment regimens, PIs with low-dose RTV (300/100 mg) are administered in combination with nucleoside reverse transcriptase inhibitors such as abacavir, lamivudine, tenofovir, and/or emtricitabine (van Heeswijk et al., 2001). Ritonavir-boosted atazanavir (ATV/r) may also be used alone as maintenance therapy (Achenbach et al., 2011; Tsai et al., 2017).
While drug boosting is an important aspect of HIV-1 therapeutic regimens, the means to study such drug-drug interactions are limited since the enzyme pathways and regulation are commonly human specific (Martignoni et al., 2006; Gautam et al., 2014). Human and mouse PXR and CAR show significant homology in CYP3A binding sites; however, homology in their ligand-binding domains is limited (Xie et al., 2000; Owen and Curley, 2015; Yan and Xie, 2016). Consequently, there are significant species differences in ligand activation of PXR and CAR and induction of CYP3A (Xie et al., 2000; Gibson et al., 2002).
We sought to modify the humanized mouse model in ways that would permit examination of human liver drug metabolizing enzymes and regulatory proteins, broadening its utility beyond studies of virus-cell-tissue interactions and ARV pharmacokinetics. To these ends, we generated a transgenic mouse with targeted replacement of mouse to human PXR, CAR, and CYP3A4/7 genes (Taconic model 11585, or PXR-CAR-CYP3A4/3A7, abbreviated hCYP3A) on a CIEA NOG mouse background. The created mice were used to evaluate the impact of human CYP3A metabolism on the pharmacokinetics of nanoformulated ATV/r (nanoATV/r) treatment (Roy et al., 2012). Plasma and liver ATV concentrations following treatment with nanoATV/r were 2- to 4-fold greater than in control mice. Cotreatment with nanoATV/r provided an approximate 3-fold enhancement of plasma and liver ATV concentrations over nanoformulated ATV (nanoATV) alone. Clear sex-dependent differences were observed for the plasma area under the concentration-time curve (AUC) and liver ATV concentrations in the nanoATV/r-treated transgenic mice. The plasma ATV AUC was 1.3-fold higher in female mice and liver ATV concentration was 1.9-fold higher in males. Taken together, a mouse model that contains the human PXR, CAR, and CYP3A4/7 genes on a genetic background suitable for humanization could be employed for studies of drug-drug interactions (Merry et al., 1997; Riska et al., 1999; van Waterschoot et al., 2010; Dellamonica et al., 2012; Holmstock et al., 2013; Scheer and Roland Wolf, 2013). These mice show clear applications for drug development and translation.
Materials and Methods
Materials.
Optima and liquid chromatography (LC)/mass spectrometry (MS) grade solvents were obtained from Fisher Scientific (Waltham, MA). Poloxamer 407 was purchased from Sigma-Aldrich (St. Louis, MO). ATV-sulfate, purchased from Gyma Laboratories of America Inc. (Westbury, MA) was free-based using triethylamine. Free-based RTV was purchased from Shengda Pharmaceutical Co. (Zhejiang, China).
Generation and Characterization of hCYP3A-NOG Mice.
Humanized PXR-CAR-CYP3A4/3A7 (hCYP3A) (C57BL/6-Nr1i3tm1(NR1I3)ArteIs(5CYP3A4-CYP3A7;Del5Cyp3a57-Cyp3a59)2ArteNr1i2tm1(NR1I2)Arte) and NOG mice were obtained from Taconic Biosciences (Hudson, NY). Mice were kept in microisolator cages with free access to food and water. Light cycles were 12:12 hour light/dark with the light phasing starting at 6:00 AM. Temperature and humidity were maintained between 21°C and 23°C and 45% and 65%, respectively. The hCYP3A strain was backcrossed to the NOG strain through a marker-assisted speed congenics protocol established in our laboratory (Gurumurthy et al., 2015). Genotypes of the mice were identified using standard polymerase chain reaction (PCR)–based genotyping reactions as previously described (Harms et al., 2014; Quadros et al., 2016). Briefly, either tail or ear piece DNA was collected from each individual animal and put into a tube containing 300 µl cell lysis buffer and 20 µl proteinase K (20 mg/ml). The mixture was kept overnight in a dry bath set at 65°C. DNA was extracted using the Qiagen Gentra Puregene Tissue Kit (Qiagen Sciences, Germantown, MD) and stored at 4°C for future use. For PCR reaction, 2× GoTaq Green Master Mix (Promega Life Sciences, Madison, WI) was diluted to 1× with nuclease free water, and 1 μl forward primer and 1 μl reverse primer (100 μM stocks) per sample were added for 100 μl master mix to a 2 ml microcentrifuge tube; 15 μl of master mix per sample and 1 μl of genomic DNA was added to the PCR tubes. The samples were then placed in a Bio-Rad T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) for amplification. Amplified products were run on a 1.5% agarose gel and analyzed via a Kodak Gel Logic 212 Imaging system (Eastman Kodak Co., Rochester, NY). Five separate PCR reactions are required to properly genotype humanized PXR-CAR-CYP3A4/3A7 mice (Supplemental Fig. 1).
A duplex PCR reaction, containing three primers, could be used for identifying both the mutant and wild-type alleles for CAR mutation, whereas two separate PCRs were needed to identify mutant and wild-type alleles for the PXR and CYP34A4 alleles. At the end of the speed congenics breeding, the heterozygous offspring were intercrossed to obtain homozygous mutations for all three loci. The primer sets used for genotyping are listed in Supplemental Table 1.
Ritonavir-Boosted Atazanavir Nanoformulations.
Poloxamer 407-coated nanoATV and nanoformulated RTV (nanoRTV) were prepared by high-pressure homogenization and characterized for size, size distribution, and zeta potential as previously described (Balkundi et al., 2011; Puligujja et al., 2013). The nanosuspensions were lyophilized and drug loading was determined by reversed-phase high-performance LC (Nowacek et al., 2009). The lyophilized drug nanosuspensions were resuspended in phosphate-buffered saline and injected intramuscularly in a total injection volume of 40 µl per 25 g mouse. Injection solutions were prepared fresh, just prior to use on treatment days. To ensure adequate homogeneous suspension, the suspensions were sonicated for 10 seconds prior to injection and vortexed frequently during treatment of animals. Drug content in the injection solutions was determine by ultra-performance LC (UPLC) tandem MS as described previously (Huang et al., 2011).
Pharmacokinetics and Drug Metabolism.
Animal studies were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals mandated by the U.S. National Institutes of Health (Bethesda, MD; https://www.ncbi.nlm.nih.gov/books/NBK54050/) and the University of Nebraska Medical Center Institutional Animal Care and Use Committee. Male and female mice, 3 to 4 months of age, were used.
The newly developed transgenic mice (hCYP3A-NOG) and wild-type (NOG) mice were treated intramuscularly with nanoATV alone (50 mg/kg) or a combination of nanoATV and nanoRTV (nanoATV/r) (20 or 50 mg/kg each drug) on days 0, 3, 7, 14, and 21. The numbers of male and female mice (N ≥ 3) in each treatment group were the same for each strain (hCYP3A-NOG and NOG). Serial blood samples were collected on days 1, 3, 7, 14, 21, and 28 into heparinized tubes and plasma prepared. Tissues were collected on day 28 (Fig. 1).
Plasma and Tissue Collection.
Fifty microliters of whole blood was collected into heparinized tubes by submandibular puncture on days 1, 3, 7, 14, and 21 (prior to injection of next dose) and 1 ml by cardiac puncture on the day of sacrifice. The blood samples were centrifuged at 3000g and plasma was collected. The plasma samples were stored at −80°C until analysis. Tissues were collected at the time of animal sacrifice. Half of each tissue was flash frozen in liquid nitrogen and stored at −80°C for drug quantitation and gene expression. The other half was fixed in 10% neutral buffered formalin for histopathology.
Quantitation of ATV and RTV.
ATV and RTV concentrations in plasma and liver were determined by UPLC tandem MS as previously described with minor modifications (Huang et al., 2011). Briefly, 100 mg liver was homogenized in four volumes of LC/MS-grade water. One milliliter of ice-cold Optima-grade acetonitrile was added to 25 µl of plasma or 100 µl liver homogenate prespiked with 10 µl internal standard (2.0 µg/ml lopinavir, 200 ng/ml final concentration). Samples were vortexed continuously for 3 minutes and centrifuged at 16,000g for 10 minutes at 4°C. The supernatants were collected and evaporated under vacuum at room temperature and then reconstituted in 100 µl of 50% Optima-grade methanol in water and sonicated for 5 minutes. After centrifugation (16,000g; 10 minutes; 4°C), 10 µl of each sample was analyzed by UPLC tandem MS for drug quantitation using a Waters Acquity H-class UPLC coupled to a Waters Xevo TQ-S Micro Triple Quadrupole Mass Spectrometer (Waters, Milford, MA). Chromatographic separation was achieved on a Waters Acquity BEH Shield RP 18 column (1.7 µm, 100 × 2.1 mm) affixed with an Acquity UPLC C18 guard column. The mobile phase consisted of (A) 5% Optima-grade acetonitrile in methanol and (B) 7.5 mM ammonium acetate adjusted to pH 4.0 with glacial acetic acid at a flow rate of 0.28 ml/min. Samples were eluted using a gradient of 70% A for 5.0 minutes, increased to 90% A over 1.0 minutes, held at 90% A for 0.8 minutes, reset to 70% A over 1.0 minutes, and then held for 1.0 minutes for column re-equilibration. ATV and RTV were quantitated using drug-to-internal-standard peak area ratios and drug calibration curves of 0.2–2000 ng/ml. ATV, RTV, and lopinavir were detected using multiple reactions monitoring transitions of 705.24 > 168.065, 721.14 > 296.104, and 629.177 > 447.202, respectively.
Gene Expression Analyses.
For relative gene expression, livers from hCYP3A-NOG and NOG mice were homogenized and total RNA was extracted using RNeasy Plus Universal Kit (Qiagen Sciences) following the manufacturer’s instructions. An equal amount of RNA (1 µg) was used to reverse transcribe into cDNA using a cDNA synthesis kit (ThermoFisher Scientific, Waltham, MA) in 40 µl reaction volume. Real-time PCR was performed on a StepOne Plus PCR (Applied Biosystems, Foster City, CA) in a singleplex 20 µl mix with human-specific (PXR, Hs00243666_m1; CAR, Hs00231959_m1; and CYP3A4, Hs00604506_m1) and mouse-specific (mPXR, Mm00803092_m1; Cyp3a11, Mm00731567_m1; mCAR, Mm01170117_m1 and Actb, Mm00607939_s1) primer-probe sets and Taqman gene expression master mix (Applied Biosystems) using 1 µl cDNA. Real-time PCR settings were as follows: 50°C for 2 minutes, and then 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute (Dagur et al., 2018). Fluorescence intensity was measured at each change of temperature to monitor amplification. Target gene expression was determined using the comparative threshold cycle method and normalized to an endogenous mouse β-actin. Fold increase in gene expression was calculated as a ratio of normalized expression in drug-treated to untreated animals.
Histopathology.
At the study end, liver tissue samples were fixed with 10% neutral buffered formalin and embedded in paraffin. Five-micron-thick sections were stained with hematoxylin and eosin. Histopathology was evaluated according to the guidelines of the Society of Toxicologic Pathology (Reston, VA; https://focusontoxpath.com/hepatotoxicity/).
Statistical Analyses.
The plasma AUC and statistical differences were determined using GraphPad Prism 7.0 (GraphPad Software Inc., LaJolla, CA). Statistical differences between the AUCs for different groups were determined using Student’s t test. Statistical differences between tissue drug concentrations were determined using one-way analysis of variance with Tukey’s multiple comparisons tests. Differences in male and female tissue drug concentrations were determined using two-way ANOVA with uncorrected Fisher’s LSD. Differences in mRNA induction of CYP3A, PXR, and CAR genes were determined by one-tailed Student’s t test. The criterion for statistical significance was P < 0.05.
Results
Generation and Characterization of the hCYP3A-NOG Mice.
Homozygous hCYP3A-NOG transgenic mice were viable and fertile with no differences from the parent NOG strain. However, hCYP3A-NOG males and females showed lower body weight compared with NOG mice (Supplemental Fig. 2). Drug treatment induced a mild reduction of parent NOG mice body weight by the end of the observation (Supplemental Figs. 2 and 3). The effect was most pronounced in NOG males treated with 50 mg/kg nanoATV/r. Importantly, no drug-related histopathological changes were observed in the livers of the mice (Supplemental Fig. 4) following nanoATV/r treatment.
RTV Affects ATV Plasma and Liver Concentrations in hCYP3A-NOG Mice.
RTV is used to boost plasma ATV concentrations in patients through inhibition of CYP3A activity (Cooper et al., 2003; Arya et al., 2012). Previous studies in our laboratory have demonstrated that weekly administration of nanoATV/r to wild-type mice (Balb/cJ, C57Bl/6, NOD scid gamma mice) provided sustained plasma ATV concentrations (Dash et al., 2012; Gautam et al., 2013, 2014). However, neither mice nor nonhuman primates completely reflected drug boosting (Martignoni et al., 2006; Gautam et al., 2014).
To determine the effect of an RTV boost, hCYP3A-NOG and NOG mice were treated weekly with nanoATV/r and plasma drug concentrations were monitored over 28 days. As shown in Fig. 2A, ATV plasma concentrations were 2- to 5-fold higher in hCYP3A-NOG mice compared with NOG mice in all treatment groups. The difference (4- to 5-fold) was most pronounced at the higher dose (50 mg/kg) at days 7–28. The AUC for plasma ATV in animals treated with 50 mg/kg nanoATV/r was 4.2-fold higher in hCYP3A-NOG mice compared with NOG mice (Table 1). In contrast, RTV plasma concentrations and AUCs were similar in both mouse strains (Fig. 2B; Table 1). RTV boosted ATV concentrations to a greater extent in hCYP3A-NOG mice than NOG mice. RTV boosted plasma ATV concentrations at day 28 by 3-fold in hCYP3A-NOG mice compared with 2-fold in NOG mice. The AUC for 50 mg/kg nanoATV/r was 2.7-fold greater than the AUC for 50 mg/kg nanoATV (19430 vs. 7111) in the transgenic mice, whereas the difference in wild-type mice was 1.9-fold (4590 vs. 2402) (Table 1). Interestingly, the ATV plasma concentrations on days 1–21 were 1.4- to 1.8-fold higher in hCYP3A-NOG females given 50 mg/kg nanoATV/r compared with males given the same treatment (Fig. 3A), which translated into an increased AUC (P = 0.04) in females (Table 1). This sex difference was not observed in NOG mice. No sex differences were observed in either strain for plasma RTV concentrations (Fig. 3B; Table 1).
The differences in plasma ATV concentrations between the mouse strains reflected differences in liver ATV concentrations. As shown in Fig. 4A, liver ATV concentrations were higher in hCYP3A-NOG mice compared with NOG mice following treatment with either 20 mg/kg (1.9-fold, 667.1 vs. 338.4 ng/g) or 50 mg/kg (3.2-fold, 3225 vs. 1002 ng/g) nanoATV/r. Inclusion of RTV boosted ATV liver concentrations by 3.2-fold (P < 0.05) in hCYP3A-NOG mice but not NOG mice. RTV liver concentrations were significantly higher (2-fold) in hCYP3A-NOG mice compared with NOG mice treated with 50 mg/kg nanoATV/r (Fig. 4B).
Liver ATV concentrations were significantly higher (1.9-fold) in hCYP3A-NOG males compared with females treated with 50 mg/kg nanoATV/r (4065 vs. 2105 ng/g; P < 0.001) (Fig. 5A). No significant sex differences were observed in mice treated with 20 mg/kg nanoATV/r or nanoATV alone. In contrast, no sex differences in liver ATV concentrations were observed in NOG mice treated with either 20 or 50 mg/kg nanoATV/r. Liver RTV concentrations were also significantly higher in hCYP3A-NOG males compared with females treated with 50 mg/kg nanoATV/r, while no differences were observed in RTV liver concentrations between NOG males and females (Fig. 5B).
Expression of CYP3A, PXR, and CAR in Livers of hCYP3A-NOG and NOG Mice.
Differences in expression of human and mouse CYP3A, PXR, and CAR genes were determined in livers of mice treated with nanoATV alone or nanoATV/r. Induction (fold increase over control) of mRNA expression for human (hCYP3A-NOG) and mouse (NOG) CYP3A4/7, PXR, and CAR was determined by real-time PCR. Induction of CYP3A4, PXR, and CAR by nanoATV and nanoATV/r was greater in both male and female hCYP3A-NOG mice compared with NOG mice (Fig. 6). CYP3A4 induction was 0.5- to 14-fold in NOG females compared with 80- to 113-fold in replicate hCYP3A-NOG mice. Responses differed in male mice compared with female mice. Induction of CYP3A4 and PXR was moderately higher in hCYP3A-NOG mice compared with NOG mice with nanoATV/r treatment. However, in contrast to the response in females, CAR induction was much greater in NOG males compared with hCYP3A-NOG males. The expression of CYP3A7, known to be expressed in human fetal liver but not adult liver (Lacroix et al., 1997; Pang et al., 2012; Betts et al., 2015), was very low in our adult mouse livers and was not induced by drug treatments (data not shown).
Discussion
Our laboratories have developed a new NOG strain of mouse containing the human PXR, CAR, and CYP3A4/7 genes. These mice, with similar survival rates and fertility to wild-type NOG mice (data not shown), were used to assess the pharmacokinetics of weekly administered nanoATV/r. There were significant differences in ATV plasma and liver concentrations and induction of CYP3A4, PXR, and CAR between the two strains. Importantly, the observed differences in ATV plasma and liver concentrations were greater in humanized mice (2.7-fold) compared with wild-type mice (1.9-fold). Cotreatment with nanoRTV boosted plasma ATV concentrations in both male and female mice (Fig. 2). These results were similar to our previous studies wherein weekly subcutaneous or intramuscular administration of nanoATV and nanoRTV provided sustained plasma ATV levels in Balb/cJ mice (Dash et al., 2012; Roy et al., 2012; Gautam et al., 2014; Puligujja et al., 2015a,b), which were boosted by cotreatment with nanoRTV. Furthermore, preliminary studies using the Taconic model 11585 (PXR-CAR-CYP3A4/3A7) C57Bl/6 background mice treated with weekly doses of nanoATV/r showed 5-fold higher ATV plasma concentrations at study end compared with wild-type mice (unpublished data). The RTV boost observed in the new strain of mice was equivalent to that observed in wild-type C57Bl/6 mice (Gautam et al., 2014). Importantly, no evidence of necrosis or acute inflammation was observed in either hCYP3A-NOG or NOG mice in the current study, which mirrored our previous observations (Dash et al., 2012; Gautam et al., 2014).
Differences in male and female responses to ARVs are well known in humans (Ofotokun, 2005) and may be associated with sex-related differences in regulation of drug metabolizing enzymes and transporters (Ofotokun, 2005). In the present study, sex differences in ATV and RTV plasma concentrations were observed in hCYP3A-NOG mice, with higher plasma ATV concentrations in females compared to males given 50 mg/kg nanoATV/r. In addition, the RTV boosting effects were 2-fold greater in female humanized mice (2.7-fold) compared with female wild-type mice (1.3-fold). Higher plasma ATV concentrations in female hCYP3A-NOG mice reflect sex differences in ATV concentrations reported in humans during treatment (Ofotokun and Pomeroy, 2003). These differences in turn may be related to a lower risk of viral control failure in women compared with men, but a higher rate of treatment discontinuation (Svedhem-Johansson et al., 2013). Our data may also predict ARV toxicity profiling, as demonstrated by the association of ATV plasma concentrations exceeding that required for viral control (upper therapeutic threshold) with a higher risk of liver and kidney dysfunction (Gervasoni et al., 2015).
The predictive value of mice with human drug metabolizing enzymes and regulatory proteins for drug-drug interaction studies have been demonstrated by a variety of other studies. The relative induction of rifampicin, sulfinpyrazone, and pioglitazone on CYP3A4 expression and pharmacokinetics of triazolam in hu-PXR-CAR-CYP3A4/7 mice was shown to be reflective of the human responses (Hasegawa et al., 2011). In another study, CYP3A-humanized mice were used to demonstrate RTV boosting of a new protease inhibitor NVS123 (Das et al., 2016). These humanized mice can also provide predictive information of the generation of human-specific drug metabolites as was shown by Nishimura et al. (2013) for clemizole in TK-NOG mice transplanted with human hepatocytes. Humanization of not only hepatic but also intestinal CYP3A is important for predicting human drug metabolites as demonstrated in studies by Nakada et al. (2016). The metabolite profile of the antidepressant drug nefazodone in murine CYP3A knockout mice with humanized livers (Cyp3a knockout chimeric mice) mirrored that observed in humans. Similarly, Barzi et al. (2017) observed human-type metabolism of ATV in PIRF mice repopulated with human hepatocytes when the murine NADPH-P450 oxidoreductase gene was deleted. The importance of CYP3A in generation of toxic ATV and RTV metabolites was shown by Li et al. (2011a,b). In immune competent C57Bl/6 background hu-PXR-CAR-CYP3A4/7 mice, differences in the metabolite profile of drugs in combination compared with normal mice have been shown for itraconazole/cobimetinib and midazolam plus the CYP3A inhibitor clarithromycin and support our observations for the interactions of ATV and RTV (Choo et al., 2015; Ly et al., 2017).
For adequate suppression of HIV replication and to reduce the development of viral drug resistance, plasma PI concentrations need to be maintained at therapeutic levels. Thus, clinically, RTV has been used to boost plasma PI concentrations (Hsu et al., 1998; Condra et al., 2000) through inhibition of CYP3A4 metabolism of PIs by intestinal epithelial cells and hepatocytes and through inhibition of the P-glycoprotein efflux transporter in intestinal epithelial cells (Holmstock et al., 2012). However, the action of RTV in humans is not straightforward. RTV inhibits CYP3A liver and intestinal activity by 70%, but can also induce CYP3A4 activity through activation of PXR (Luo et al., 2002). Using our new hCYP3A-NOG strain of mice we observed the net outcome for injectable long-acting formulations that exhibit complex interactions resulting from induction and inhibition of P450 enzymes.
The critical role of PXR and CAR in the regulation of phase I (oxidation) and phase II (conjugation) drug metabolizing enzymes and transporters, their selectivity for different ligands including endogenous lipids and hormones, their influence on inflammatory responses (Kusunoki et al., 2014), and their regulation of cholesterol, glucose, and lipid metabolism (Li et al., 2007; Timsit and Negishi, 2007; Moreau et al., 2008; Yan et al., 2015) can elicit distinct differences between mice and humans. For example, the synthetic glucocorticoid dexamethasone is a more potent inducer of Cyp3a in wild-type mice compared with mice humanized for PXR (Scheer et al., 2010). In another study, PXR and CAR humanized mice, but not wild-type mice, displayed increased microsomal protein content, total P450 content, and P450 reductase activity in response to rifampicin treatment (Lee et al., 2009). Species-specific differences in PXR and CAR activation and their regulation of energy metabolism may also underlie the differences in body weight in the hCYP3A-NOG and control NOG mice and the slight weight loss following treatment in NOG mice observed in the current study. Differences in such responses and substrate specificities can markedly influence the translation of studies for drug bioavailability, distribution, toxicity, and efficacy from mice to humans. The new humanized immune-deficient mouse strain offers the potential for studying human PXR and CAR regulation of drug metabolism and inflammatory responses in the context of drug treatment of immune-mediated disease.
Limitations of this humanized mouse model are that it does not emulate human variability of gene expressions, does not incorporate polymorphisms in drug metabolizing enzymes that influence therapeutic efficacy and toxicity, and does not incorporate humanization of drug efflux transporters such as P-glycoprotein that affect PI distribution in sanctuary sites (e.g., brain, vaginal mucosa, testes) (Griffin et al., 2011). The non-nucleoside reverse transcriptase inhibitor efavirenz can activate PXR and promote CYP3A4 activity in patients on ARV treatment (Hariparsad et al., 2004; Fellay et al., 2005), and a single nucleotide polymorphism in PXR is associated with ATV plasma trough levels below the minimum effective concentration (Siccardi et al., 2008). Furthermore, while these mice are useful in examining the human/mouse differences in PXR and CAR regulation and CYP3A4 metabolism of ARVs, other drug metabolizing enzymes and transporters also contribute to the bioavailability and pharmacokinetics of ARVs. As an example, ATV is a substrate for CYP3A5, P-glycoprotein, multidrug resistance protein 1, and human organic anion transporters, and an inhibitor of P-glycoprotein and breast cancer resistance protein (Bousquet et al., 2008; Janneh et al., 2009; Achenbach et al., 2011). CYP2D6, with well-known poor and extensive metabolizer polymorphism, can contribute variably to the metabolism of RTV (Kumar et al., 1996; Hsu et al., 1998; Kaspera et al., 2014). In addition, RTV induces CYP2B6, which contributes to the metabolism of PIs as well as efavirenz (Dixit et al., 2007; Foisy et al., 2008). Thus, species differences and polymorphisms in substrate specificities and activities in these enzymes and transporters could affect the pharmacokinetics and drug-drug interactions observed for ARVs in this humanized mouse model.
In addition, the downstream genetic and protein responses are those of the mouse, not humans. Despite these limitations, the hu-PXR-CAR-CYP3A4/7 model can provide predictive values for the development of drug combinations with reduced drug-drug interactions and toxicities. The important advantage of the new hCYP3A-NOG mouse strain is their suitability for immune reconstitution by transplantation of human hematopoietic stem cells and human tissue tumor xenografts that will provide human-like metabolism of combinations of ARV and anticancer drugs. This new strain of mice could also be used for testing drug combinations related to treatment of cancers not previously associated with HIV, such as lung, head and neck, liver, anal, and kidney cancers, which occur in HIV-infected individuals at much higher rates than in the general population (Deeken et al., 2015).
For future studies, this new strain of immune deficient mice will be used to determine the biologic activities of clinically used ARV combinations and for development of long-acting formulations of these ARVs in human CD34+ hematopoietic stem cell-reconstituted HIV-1 infected animals.
Acknowledgments
We acknowledge the technical assistance of Diana Palandri and Celina Prince.
Authorship Contributions
Participated in research design: Poluektova, McMillan, Gendelman, Gorantla, Gurumurthy.
Conducted experiments: Cobb, Lin, Banoub, Dagur, Makarov.
Contributed new reagents or analytic tools: Branch Woods, Wang, Joshi, Quadros, Harms, Gurumurthy.
Performed data analysis: Poluektova, McMillan, Dagur, Kocher, Cohen.
Wrote or contributed to the writing of the manuscript: McMillan, Poluektova, Gendelman, Gurumurthy.
Footnotes
- Received December 18, 2017.
- Accepted February 22, 2018.
This work was supported by a University of Nebraska Medical Center Graduate Student Fellowship and the National Institutes of Health [Grants R24OD018546, P01DA028555, R01NS036126, and R01AG043540].
↵This article has supplemental material available at jpet.aspetjournals.org.
Abbreviations
- ARV
- antiretroviral
- ATV
- atazanavir
- AUC
- area under the concentration-time curve
- CAR
- constitutive androstane receptor
- HIV
- human immunodeficiency virus
- HIV-1
- human immunodeficiency virus type 1
- LC
- liquid chromatography
- MS
- mass spectrometry
- nanoATV
- nanoformulated atazanavir
- nanoATV/r
- nanoformulated ritonavir-boosted atazanavir
- nanoRTV
- nanoformulated ritonavir
- P450
- cytochrome P450
- PCR
- polymerase chain reaction
- PI
- protease inhibitor
- PXR
- pregnane X receptor
- RTV
- ritonavir
- UPLC
- ultra-performance liquid chromatography
- Copyright © 2018 by The Author(s)
This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.