After 50 Years of Hepatic Clearance Models, Where Should We Go from Here? Improvements and Implications for Physiologically Based Pharmacokinetic Modeling.

There is overwhelming preference for application of the unphysiologic, well-stirred model (WSM) over the parallel tube model (PTM) and dispersion model (DM) to predict hepatic drug clearance, CLH , despite that liver blood flow is dispersive and closer to the DM in nature. The reasoning is the ease in computation relating the hepatic intrinsic clearance ( CLint ), hepatic blood flow ( QH ), unbound fraction in blood ( fub ) and the transmembrane clearances ( CLin and CLef ) to CLH for the WSM. However, the WSM, being the least efficient liver model, predicts a lower EH that is associated with the in vitro CLint ( Vmax / Km ), therefore requiring scale-up to predict CLH in vivo. By contrast, the miniPTM, a three-subcompartment tank-in-series model of uniform enzymes, closely mimics the DM and yielded similar patterns for CLint versus EH , substrate concentration [S] , and KL / B , the tissue to outflow blood concentration ratio. We placed these liver models nested within physiologically based pharmacokinetic models to describe the kinetics of the flow-limited, phenolic substrate, harmol, using the WSM (single compartment) and the miniPTM and zonal liver models (ZLMs) of evenly and unevenly distributed glucuronidation and sulfation activities, respectively, to predict CLH For the same, given CLint ( Vmax and Km ), the WSM again furnished the lowest extraction ratio ( EH,WSM = 0.5) compared with the miniPTM and ZLM (>0.68). Values of EH,WSM were elevated to those for EH, PTM and EH, ZLM when the Vmax s for sulfation and glucuronidation were raised 5.7- to 1.15-fold. The miniPTM is easily manageable mathematically and should be the new normal for liver/physiologic modeling. SIGNIFICANCE STATEMENT: Selection of the proper liver clearance model impacts strongly on CLH predictions. The authors recommend use of the tank-in-series miniPTM (3 compartments mini-parallel tube model), which displays similar properties as the dispersion model (DM) in relating CLint and [ S ] to CLH as a stand-in for the DM, which best describes the liver microcirculation. The miniPTM is readily modified to accommodate enzyme and transporter zonation.

Aging and brain free cholesterol concentration on amyloid-ß peptide accumulation in guinea pigs.

Alzheimer's disease is a complex multifactorial neurodegenerative disorder wherein age is a major risk factor. The appropriateness of the Hartley guinea pig (GP), which displays high sequence homologies of its amyloid-ß (Aß40 and Aß42) peptides, Mdr1 and APP (amyloid precursor protein) and similarity in lipid handling to humans, was appraised among 9-40 weeks old guinea pigs. Protein expression levels of P-gp (Abcb1) and Cyp46a1 (24(S)-hydroxylase) for Aß40, and Aß42 efflux and cholesterol metabolism, respectively, were decreased with age, whereas those for Lrp1 (low-density lipoprotein receptor related protein 1), Rage (receptor for advanced glycation endproducts) for Aß efflux and influx, respectively, and Abca1 (the ATP binding cassette subfamily A member 1) for cholesterol efflux, were unchanged among the ages examined. There was a strong, negative correlation of the brain Aß peptide concentrations and Abca1 protein expression levels with free cholesterol. The correlation of Aß peptide concentrations with Cyp46a1 was, however, not significant, and concentrations of the 24(S)-hydroxycholesterol metabolite revealed a decreasing trend from 20 weeks old toward 40 weeks old guinea pigs. The composite data suggest a role for free cholesterol on brain Aß accumulation. The decreases in P-gp and Lrp1 protein levels should further exacerbate the accumulation of Aß peptides in guinea pig brain.

Kirchhoff's Laws and Hepatic Clearance, Well-Stirred Model - Is There Common Ground?

Clearance concepts are extensively applied in drug development and drug therapy. The well-stirred model (WSM) of hepatic elimination is the most widely adopted physiologic model in pharmacokinetics owing to its simplicity. A common feature of this organ model is its use to relate hepatic clearance of a compound to the physiologic variables: organ blood flow rate, binding within blood, and hepatocellular metabolic and excretory activities. Recently, Kirchhoff's laws of electrical network have been applied to organ clearance (Pachter et al., 2022; Benet and Sodhi, 2023) with the claim that they yield the same equation for hepatic clearance as the WSM, and that the equation is independent of a mechanistic model. This commentary analyzes this claim and shows that implicit in the application of Kirchhoff's approaches are the same assumptions as those of the WSM. Concern is also expressed in the interpretation of permeability or transport parameters and related equations, as well as the inappropriateness of the corresponding equation defining hepatic clearance. There is no value, and some dangers, in applying Kirchhoff's electrical laws to organ clearance. SIGNIFICANCE STATEMENT: This commentary refutes this claim by Pachter et al. (2022), and Benet and Sodhi, (2023), who suggest that the well-stirred model (WSM) of hepatic elimination, the most widely applied physiologic model of hepatic clearance, provides the same equation as Kirchhoff's laws of electrical network that is independent of a physiologic model. A careful review shows that the claim is groundless and fraught with errors. We conclude that there is no place for the application of Kirchhoff's laws to organ clearance models.

In Defense of Current Concepts and Applications of Clearance in Drug Development and Therapeutics.

Clearance is one of the most widely quoted and applied pharmacokinetic concepts in drug development and therapy. Its foundations and associated models of drug elimination are well embedded and accepted within the scientific community. Recently, however, the prevailing views that have held us in good stead for the past almost 50 years have been challenged with the argument that organ clearance should not be based on elimination rate, now defined by extraction across the liver divided by incoming or systemic concentration, as in current practice, but rather, by the mean concentration of drug within the blood in the organ, which is model-dependent. We argue that all needed parameters already exist, and that the proposed new approach to organ clearance is confusing and unnecessary. SIGNIFICANCE STATEMENT: Clearance concepts are widely applied in drug development and therapy. Historically, hepatic clearance has been defined as the ratio of rate of elimination divided by ingoing blood concentration. Recently, this approach has been challenged arguing that clearance should be referenced to blood concentration within the liver extrapolation (IVIVE). There is no need for additional, a feature that corresponds to intrinsic clearance of the chosen clearance model, a widely accepted parameter in physiologically based pharmacokinetic (PBPK) and in vitro to in vivo extrapolation (IVIVE). There is no need for additional, confusing clearance terms, which offer no material benefit.

Impact of age, hypercholesterolemia, and the vitamin D receptor on brain endogenous ß-amyloid peptide accumulation in mice.

Age, hypercholesterolemia, and vitamin D deficiency are risk factors that increase the brain accumulation of pathogenic ß-amyloid peptides (40 and 42), precursors leading to Alzheimer's disease (AD) in humans. The relative changes accompanying aging, high cholesterol, and/or treatment of calcitriol, active vitamin D receptor (VDR) ligand, under normal physiology are unknown. We examined these relative changes in C57BL/6 mice of ages 2, 4-8, and more than 10 months old, which were fed a normal or high fat / high cholesterol diet and treated with calcitriol, active ligand of the vitamin D receptor (0 or 2.5 µg/kg ×4, intraperitoneally, every other day to elicit cholesterol lowering in liver). Aß40 but not Aß42 accumulation in brain and lower P-glycoprotein (P-gp) and neprilysin protein expressions for Aß efflux and degradation, respectively, were found to be associated with aging. But there was no trend for BACE1 (ß-secretase 1, a cholesterol-sensitive enzyme) toward Aß synthesis with age. In response to calcitriol treatment, P-gp was elevated, mitigating partially the age-related changes. Although age-dependent decreasing trends in mRNA expression levels existed for Cyp46a1, the brain cholesterol processing enzyme, whose inhibition increases BACE1 and ApoE to facilitate microglia Aß degradation, mRNA changes for other cholesterol transporters: Acat1 and Abca1, and brain cholesterol levels remained unchanged. There was no observable change in the mRNA expression of amyloid precursor protein (APP) and the influx (RAGE) and efflux (LRP1) transporters with respect to age, diet, or calcitriol treatment. Overall, aging poses as a risk factor contributing to Aß accumulation in brain, and VDR-mediated P-gp activation partially alleviates the outcome.

Human Amyloid-ß40 Kinetics after Intravenous and Intracerebroventricular Injections and Calcitriol Treatment in Rats In Vivo.

Amyloid-ß peptides of 40 and 42 amino acid lengths, which are synthesized in neurons and degraded in the brain and liver, have the potential to aggregate and form neuritic plaques in Alzheimer disease. The kinetics of human amyloid-ß (hAß) 40 were examined in the rat pursuant to intravenous and intracerebroventricular administration after pretreatment with calcitriol, the active vitamin D receptor ligand (6.4 nmol·kg-1 in 0.3 ml corn oil every other day for four intraperitoneal doses) to induce P-glycoprotein (P-gp) and enhance hAß40 brain efflux. The interference of hAß40 by media matrix that suppressed absorbance readings in the ELISA assay was circumvented with use of different calibration curves prepared in Standard Dilution Buffer, undiluted, 10-10,000 or 5-fold diluted plasma, or artificial cerebrospinal fluid. Simultaneous fitting of hAß40 plasma and cerebrospinal fluid (CSF) data after intravenous and intracerebroventricular administration were described by catenary-mammillary models comprising of a central and two peripheral compartments, the brain, and one to four CSF compartments. The model with only one CSF compartment (model I) best fitted the intravenous data that showed a faster plasma decay t1/2 and slower equilibration between plasma and brain/CSF. Calcitriol induction increased the brain efflux rate constant, k41 (1.8-fold), at the blood-brain barrier when compared with the control group, as confirmed by the 2-fold (P < 0.05) increase in brain P-gp relative protein expression. SIGNIFICANCE STATEMENT: An accurate description of the kinetic behavior of human amyloid-ß (hAß) 40 is needed in defining the toxic peptide as a biomarker of Alzheimer disease. Modeling of hAß40 data after intravenous and intracerebroventricular administration to the rat revealed an initially faster plasma half-life that reflected faster peripheral distribution but slower equilibration between plasma and brain/cerebrospinal fluid even with calcitriol pretreatment that increased P-glycoprotein protein expression and enhanced efflux clearance from brain.

Current Evidence and Future Directions of Tranexamic Acid Use, Efficacy, and Dosing for Major Surgical Procedures.

Tranexamic acid reduces blood loss and transfusion requirements with no significant thrombotic adverse effects. Postoperative seizures have been seen in cardiac surgical patients in association with patient (advanced age, underlying neurologic disease, chronic kidney disease); surgical (open cardiac procedures, long bypass times); and drug (high tranexamic acid dose) risk factors. Tranexamic acid dosing regimens should be decreased in patients with chronic kidney dysfunction secondary to reduced clearance and drug accumulation. Optimal dosing for cardiac surgical patients has been recommended. Additional research is required to determine dosing regimens in major noncardiac surgery and plasma concentration levels associated with inducing seizures.

Noteworthy idiosyncrasies of 1α,25-dihydroxyvitamin D3 kinetics for extrapolation from mouse to man: Commentary.

Calcitriol or 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] is the active ligand of the vitamin D receptor (VDR) that plays a vital role in health and disease. Vitamin D is converted to the relatively inactive metabolite, 25-hydroxyvitamin D3 [25(OH)D3 ], by CYP27A1 and CYP2R1 in the liver, then to 1,25(OH)2 D3 by a specific, mitochondrial enzyme, CYP27B1 (1α-hydroxylase) that is present primarily in the kidney. The degradation of both metabolites is mostly carried out by the more ubiquitous mitochondrial enzyme, CYP24A1. Despite the fact that calcitriol inhibits its formation and degradation, allometric scaling revealed strong interspecies correlation of the net calcitriol clearance (CL estimated from dose/AUC∞ ), production rate (PR), and basal, plasma calcitriol concentration with body weight (BW). PBPK-PD (physiologically based pharmacokinetic-pharmacodynamic) modeling confirmed the dynamic interactions between calcitriol and Cyp27b1/Cyp24a1 on the decrease in the PR and increase in CL in mice. Close scrutiny of the literature revealed that basal levels of calcitriol had not been taken into consideration for estimating the correct AUC∞ and CL after exogenous calcitriol dosing in both animals and humans, leading to an overestimation of AUC∞ and underestimation of the plasma CL. In humans, CL was decreased in chronic kidney disease but increased in cancer. Collectively, careful pharmacokinetic data analysis and improved definition are achieved with PBPK-PD modeling, which embellishes the complexity of dose, enzyme regulation, and disease conditions. Allometric scaling and PBPK-PD modeling were applied successfully to extend the PBPK model to predict calcitriol kinetics in cancer patients.

Theoretical consideration of the properties of intestinal flow models on route-dependent drug removal: Segregated Flow (SFM) vs. Traditional (TM).

The intestine is endowed with a plethora of enzymes and transporters and regulates the flow of substrate to the liver. Physiologically-based pharmacokinetic models have surfaced to describe intestinal removal. The traditional model (TM) describes the intestinal flow as a whole perfusing the entire tissue that contains the intestinal transporters and enzymes. The segregated flow model (SFM) describes that only a fraction (fQ  < 0.2) of the intestinal blood flow perfuses the enterocyte region where the intestinal enzymes and transporters are housed, rendering a lower drug distribution/intestinal clearance when drug enters via the circulation than from the gut lumen. As shown by simulations, a higher intestinal clearance and extraction ratio (EI,iv ) exists for the TM than for SFM after iv dosing. By contrast, the EI,po after po dosing is higher for the SFM, due to the smaller volume of distribution for the enterocyte region and a lower flow rate that result in increased mean residence time and higher drug extraction. Under MBI (mechanism-based inhibition), the AUCR,po after oral bolus is the highest for drug when inhibitor is given orally, with SFM > TM. Competitive inhibition of intestinal enzymes leads to higher liver metabolism; again, when both drug and inhibitor are given orally, changes in the SFM > TM. However, less definitive patterns result with inhibition of both intestinal and liver enzymes. In conclusion, differences exist for EI and drug-drug interaction (DDI) between the TM and SFM. The fractional intestinal blood flow (fQ ) is a key factor affecting different extents of intestinal/liver metabolism of the drug after oral as well as intravenous administration.

Finding Tmax and Cmax in Multicompartmental Models.

Drug absorption data are critical in bioequivalence comparisons, and factors such as the maximum drug concentration (Cmax), time to achieve Cmax (or Tmax), as well as the area under the curve (AUC) are important metrics. It is generally accepted that the AUC is a meaningful estimate of the extent of absorption, and Tmax or Cmax may be used for assessing the rate of absorption. But estimation of the rate of absorption with Tmax or Cmax is not always feasible, as explicit solutions relating Tmax and Cmax to the absorption (ka) and elimination rate (k) constants exist only for the one and not multicompartmental oral model. Therefore, the determination of Tmax or Cmax for multicompartmental models is uncertain. Here, we propose an alternate, numerical approach that uses the point-slope method for the first and second derivative(s) of the concentration-versus-time profiles and the Newton-Raphson iteration method for the determination of Tmax and Cmax We show that the method holds for multicompartmental oral dosing under single or steady-state conditions in the absence of known microconstants, even for flip-flop (ka < ß) models. Simulations showed that the Cmax and Tmax estimates obtained with the Newton-Raphson method were more accurate than those based on the noncompartmental, observation-based method recommended by the US Food and Drug Administration. The %Bias attributable to sampling frequency and assay error were less than those determined by the noncompartmental method, showing that the Newton-Raphson method is viable for the estimation of Tmax and Cmax.

Highlighting Vitamin D Receptor-Targeted Activities of 1α,25-Dihydroxyvitamin D3 in Mice via Physiologically Based Pharmacokinetic-Pharmacodynamic Modeling.

We expanded our published physiologically based pharmacokinetic model (PBPK) on 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], ligand of the vitamin D receptor (VDR), to appraise VDR-mediated pharmacodynamics in mice. Since 1,25(OH)2D3 kinetics was best described by a segregated-flow intestinal model (SFM) that described a low/partial intestinal (blood/plasma) flow to enterocytes, with feedback regulation of its synthesis (Cyp27b1) and degradation (Cyp24a1) enzymes, this PBPK(SFM) model was expanded to describe the VDR-mediated changes (altered/basal mRNA expression) of target genes/responses with the indirect response model. We examined data on 1) renal Trpv5 (transient receptor potential cation channel, subfamily V member 5) and Trpv6 and intestinal Trpv6 (calcium channels) for calcium absorption; 2) liver 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (Hmgcr) and cytochrome 7α-hydroxylase (Cyp7a1) for cholesterol synthesis and degradation, respectively; and 3) renal and brain Mdr1 (multidrug-resistance protein that encodes the P-glycoprotein) for digoxin disposition after repetitive intraperitoneal doses of 120 pmol 1,25(OH)2D3 Fitting, performed with modeling software, yielded reasonable prediction of a dominant role of intestinal Trpv6 in calcium absorption, circadian rhythm that is characterized by simple cosine models for Hmgcr and Cyp7a1 on liver cholesterol, and brain and renal Mdr1 on tissue efflux of digoxin. Fitted parameters on the Emax, EC50, and turnover rate constants of VDR-target genes [zero-order production (kin) and first-order degradation (kout) rate constants] showed low coefficients of variation and acceptable median prediction errors (4.5%-40.6%). Sensitivity analyses showed that the Emax and EC50 values are key parameters that could influence the pharmacodynamic responses. In conclusion, the PBPK(SFM)-pharmacodynamic model successfully characterized VDR gene activation and serves as a useful tool to predict the therapeutic effects of 1,25(OH)2D3.

Tranexamic Acid Dosing for Cardiac Surgical Patients With Chronic Renal Dysfunction: A New Dosing Regimen.

BACKGROUND: Tranexamic acid (TXA) is a common antifibrinolytic agent used to minimize bleeding in cardiac surgery. Up to 50% cardiac surgical patients have chronic renal dysfunction (CRD). Optimal dosing of TXA in CRD remains poorly investigated. This is important as TXA is renally eliminated with accumulation in CRD. High TXA doses are associated with postoperative seizures. This study measures plasma TXA concentrations in CRD cardiac surgical patients for pharmacokinetic modeling and dose adjustment recommendations. METHODS: This prospective cohort study enrolled 48 patients with stages 1-5 CRD, classified by Kidney Disease Outcome Quality Initiative. Patients were separated into 2 treatment groups. A "low-risk" group underwent simple aortocoronary bypass or single-valve repair/replacement and received a 50 mg/kg TXA bolus. A "high-risk" group underwent redo, aortic, multiple valve or combination surgery and received the Blood Conservation Using Anti-fibrinolytics Trial dosing regimen (loading dose 30 mg/kg, infusion 16 mg/kg/h with 2 mg/kg in pump prime). Primary outcome identified changes in TXA clearance and distribution volume, which provided the rationale for dose adjustment. Descriptive clinical outcomes assessed postoperative seizures, blood loss, ischemic-thrombotic complications, in-hospital mortality, and length of hospital stay. RESULTS: TXA concentrations were elevated and sustained above the therapeutic threshold for approximately 12 hours in high-risk stages 3-5 groups, in accordance to CRD severity. CONCLUSIONS: Using a pharmacokinetic model, we propose a simple new TXA dosing regimen that optimizes maximal antifibrinolysis and avoids excessive drug dosing.

Potencies of vitamin D analogs, 1α-hydroxyvitamin D3 , 1α-hydroxyvitamin D2 and 25-hydroxyvitamin D3 , in lowering cholesterol in hypercholesterolemic mice in vivo.

Vitamin D3 and the synthetic vitamin D analogs, 1α-hydroxyvitamin D3 [1α(OH)D3 ], 1α-hydroxyvitamin D2 [1α(OH)D2 ] and 25-hydroxyvitamin D3 [25(OH)D3 ] were appraised for their vitamin D receptor (VDR) associated-potencies as cholesterol lowering agents in mice in vivo. These precursors are activated in vivo: 1α(OH)D3 and 1α(OH)D2 are transformed by liver CYP2R1 and CYP27A1 to active VDR ligands, 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] and 1α,25-dihydroxyvitamin D2 [1,25(OH)2 D2 ], respectively. 1α(OH)D2 may also be activated by CYP24A1 to 1α,24-dihydroxyvitamin D2 [1,24(OH)2 D2 ], another active VDR ligand. 25(OH)D3 , the metabolite formed via CYP2R1 and or CYP27A1 in liver from vitamin D3 , is activated by CYP27B1 in the kidney to 1,25(OH)2 D3 . In C57BL/6 mice fed the high fat/high cholesterol Western diet for 3 weeks, vitamin D analogs were administered every other day intraperitoneally during the last week of the diet. The rank order for cholesterol lowering, achieved via mouse liver small heterodimer partner (Shp) inhibition and increased cholesterol 7α-hydroxylase (Cyp7a1) expression, was: 1.75 nmol/kg 1α(OH)D3  > 1248 nmol/kg 25(OH)D3 (dose ratio of 0.0014) > > 1625 nmol/kg vitamin D3 . Except for 1.21 nmol/kg 1α(OH)D2 that failed to lower liver and plasma cholesterol contents, a significant negative correlation was observed between the liver concentration of 1,25(OH)2 D3 formed from the precursors and liver cholesterol levels. The composite results show that vitamin D analogs 1α(OH)D3 and 25(OH)D3 exhibit cholesterol lowering properties upon activation to 1,25(OH)2 D3 : 1α(OH)D3 is rapidly activated by liver enzymes and 25(OH)D3 is slowly activated by renal Cyp27b1 in mouse.

Alterations in gene expression in vitamin D-deficiency: Down-regulation of liver Cyp7a1 and renal Oat3 in mice.

The vitamin D-deficient model, established in the C57BL/6 mouse after 8 weeks of feeding vitamin D-deficient diets in the absence or presence of added calcium, was found associated with elevated levels of plasma parathyroid hormone (PTH) and plasma and liver cholesterol, and a reduction in cholesterol 7α-hydroxylase (Cyp7a1, rate-limiting enzyme for cholesterol metabolism) and renal Oat3 mRNA/protein expression levels. However, there was no change in plasma calcium and phosphate levels. Appraisal of the liver revealed an up-regulation of mRNA expressions of the small heterodimer partner (Shp) and attenuation of Cyp7a1, which contributed to hypercholesterolemia in vitamin D-deficiency. When vitamin D-sufficient or D-deficient mice were further rendered hypercholesterolemic with 3 weeks of feeding the respective, high fat/high cholesterol (HF/HC) diets, treatment with 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], active vitamin D receptor (VDR) ligand, or vitamin D (cholecalciferol) to HF/HC vitamin D-deficient mice lowered the cholesterol back to baseline levels. Cholecalciferol treatment partially restored renal Oat3 mRNA/protein expression back to that of vitamin D-sufficient mice. When the protein expression of protein kinase C (PKC), a known, negative regulator of Oat3, was examined in murine kidney, no difference in PKC expression was observed for any of the diets with/without 1,25(OH)2 D3 /cholecalciferol treatment, inferring that VDR regulation of renal Oat3 did not involve PKC in mice. As expected, plasma calcium levels were not elevated by cholecalciferol treatment of vitamin D-deficient mice, while 1,25(OH)2 D3 treatment led to hypercalcemia. In conclusion, vitamin D-deficiency resulted in down-regulation of liver Cyp7a1 and renal Oat3, conditions that are alleviated upon replenishment of cholecalciferol.

Disrupted Murine Gut-to-Human Liver Signaling Alters Bile Acid Homeostasis in Humanized Mouse Liver Models.

The humanized liver mouse model is being exploited increasingly for human drug metabolism studies. However, its model stability, intercommunication between human hepatocytes and mouse nonparenchymal cells in liver and murine intestine, and changes in extrahepatic transporter and enzyme expressions have not been investigated. We examined these issues in FRGN [fumarylacetoacetate hydrolase (Fah-/-), recombination activating gene 2 (Rag2-/-), and interleukin 2 receptor subunit gamma (IL-2rg -/-) triple knockout] on nonobese diabetic (NOD) background] and chimeric mice: mFRGN and hFRGN (repopulated with mouse or human hepatocytes, respectively). hFRGN mice showed markedly higher levels of liver cholesterol, biliary bilirubin, and bile acids (liver, bile, and plasma; mainly human forms, but also murine bile acids) but lower transforming growth factor beta receptor 2 (TGFBR2) mRNA expression levels (10%) in human hepatocytes and other proliferative markers in mouse nonparenchymal cells (Tgf-ß1) and cholangiocytes [plasma membrane-bound, G protein-coupled receptor for bile acids (Tgr5)], suggestive of irregular regeneration processes in hFRGN livers. Changes in gene expression in murine intestine, kidney, and brain of hFRGN mice, in particular, induction of intestinal farnesoid X receptor (Fxr) genes: fibroblast growth factor 15 (Fgf15), mouse ileal bile acid binding protein (Ibabp), small heterodimer partner (Shp), and the organic solute transporter alpha (Ostα), were observed. Proteomics revealed persistence of remnant murine proteins (cyotchrome P450 7α-hydroxylase (Cyp7a1) and other enzymes and transporters) in hFRGN livers and suggest the likelihood of mouse activity. When compared with normal human liver tissue, hFRGN livers showed lower SHP mRNA and higher CYP7A1 (300%) protein expression, consequences of tß- and tα-muricholic acid-mediated inhibition of the FXR-SHP cascade and miscommunication between intestinal Fgf15 and human liver fibroblast growth factor receptor 4 (FGFR4), as confirmed by the unchanged hepatic pERK/total ERK ratio. Dysregulation of hepatocyte proliferation and bile acid homeostasis in hFRGN livers led to hepatotoxicity, gallbladder distension, liver deformity, and other extrahepatic changes, making questionable the use of the preparation for drug metabolism studies.

Unequivocal evidence supporting the segregated flow intestinal model that discriminates intestine versus liver first-pass removal with PBPK modeling.

Merits of the segregated flow model (SFM), highlighting the intestine as inert serosa and active enterocyte regions, with a smaller fractional (fQ < 0.3) intestinal flow (QI ) perfusing the enterocyte region, are described. Less drug in the circulation reaches the enterocytes due to the lower flow (fQ QI ) in comparison with drug administered into the gut lumen, fostering the idea of route-dependent intestinal removal. The SFM has been found superior to the traditional model (TM), which views the serosa and enterocytes totally as a well-mixed tissue perfused by 100% of the intestinal flow, QI . The SFM model is able to explain the lower extents of intestinal metabolism of enalapril, morphine and midazolam with i.v. vs. p.o. dosing. For morphine, the urine/bile ratio of the metabolite, morphine glucuronide MGurineMGbile for p.o. was 2.6× that of i.v. This was due to the higher proportion of intestinally formed morphine glucuronide, appearing more in urine than in bile due to its low permeability and greater extent of intestinal formation with p.o. administration. By contrast, the TM predicted the same MGurineMGbile for p.o. vs. i.v. The TM predicted that the contributions of the intestine:liver to first-pass removal were 46%:54% for both p.o. and i.v. The SFM predicted same 46%:54% (intestine:liver) for p.o., but 9%:91% for i.v. By contrast, the kinetics of codeine, the precursor of morphine, was described equally well by the SFM- and TM-PBPK models, a trend suggesting that intestinal metabolism of codeine is negligible. Fits to these PBPK models further provide insightful information towards metabolite formation: available fractions and the fractions of hepatic and total clearances that form the metabolite in question. The SFM-PBPK model is useful to identify not only the presence of intestinal metabolism but the contributions of the intestine and liver for metabolite formation. Copyright © 2016 John Wiley & Sons, Ltd.

Physiologically based pharmacokinetic modeling revealed minimal codeine intestinal metabolism in first-pass removal in rats.

The physiologically based model with segregated flow to the intestine (SFM-PBPK; partial, lower flow to enterocyte region vs. greater flow to serosal region) was found to describe the first-pass glucuronidation of morphine (M) to morphine-3ß-glucuronide (MG) in rats after intraduodenal (i.d.) and intravenous (i.v.) administration better than the traditional model (TM), for which a single intestinal flow perfused the whole of the intestinal tissue. The segregated flow model (SFM) described a disproportionately greater extent of intestinal morphine glucuronidation for i.d. vs. i.v. administration. The present study applied the same PBPK modeling approaches to examine the contributions of the intestine and liver on the first-pass metabolism of the precursor, codeine (C, 3-methylmorphine) in the rat. Unexpectedly, the profiles of codeine, morphine and morphine-3ß-glucuronide in whole blood, bile and urine, assayed by LCMS, were equally well described by both the TM-PBPK and SFM-PBPK. The fitted parameters for the models were similar, and the net formation intrinsic clearance of morphine (from codeine) for the liver was much higher, being 9- to 13-fold that of the intestine. Simulations, based on the absence of intestinal formation of morphine, correlated well with observations. The lack of discrimination of SFM and TM with the codeine data did not invalidate the SFM-PBPK model but rather suggests that the liver is the only major organ for codeine metabolism. Because of little or no contribution by the intestine to the metabolism of codeine, both the TM- and SFM-PBPK models are equally consistent with the data. Copyright © 2016 John Wiley & Sons, Ltd.

Comparing early liver graft function from heart beating and living-donors: A pilot study aiming to identify new biomarkers of liver injury.

The liver and kidney functions of recipients of liver transplantation (LT) surgery with heart beating (HBD, n = 13) or living donors (LD, n = 9) with different cold ischemia times were examined during the neohepatic phase for the elimination of rocuronium bromide (ROC, cleared by liver and kidney) and tranexamic acid (TXA, cleared by kidney). Solid phase micro-extraction and LC-MS/MS was applied to determine the plasma concentrations of ROC and TXA, and creatinine was determined by standard laboratory methods. Metabolomics and the relative expressions of miR-122, miR-148a and γ-glutamyltranspeptidase (GGT), liver injury biomarkers, were also measured. The ROC clearance for HBD was significantly lower than that for LD (0.147 ± 0.052 vs. 0.265 ± 0.148 ml·min-1 ·g-1 liver) after intravenous injection (0.6 mg·kg-1 ). The clearance of TXA, a compound cleared by glomerular filtration, given as a 1 g bolus followed by infusion (10 mg·kg-1 ·h-1 ), was similar between HBD and LD groups (~ 1 ml·min-1 ·kg-1 ). The TXA clearance in both groups was lower than the GFR, showing a small extent of hepatorenal coupling. The miR-122 and miR-148a expressions were similar for the HBD and LD groups, whereas GGT expression was significantly increased for HBD. The lower ROC clearance and the higher GGT levels in the HBD group of longer cold ischemia times performed worse than the LD group during the neophase. Metabololmics further showed clusters of bile acids, phospholipids and lipid ω-oxidation products for the LD and HBD groups. In conclusion, ROC CL and GGT expression, and metabolomics could serve as sensitive indices of early graft function. Copyright © 2017 John Wiley & Sons, Ltd.

Metabolite Kinetics: The Segregated Flow Model for Intestinal and Whole Body Physiologically Based Pharmacokinetic Modeling to Describe Intestinal and Hepatic Glucuronidation of Morphine in Rats In Vivo.

We used the intestinal segregated flow model (SFM) versus the traditional model (TM), nested within physiologically based pharmacokinetic (PBPK) models, to describe the biliary and urinary excretion of morphine 3ß-glucuronide (MG) after intravenous and intraduodenal dosing of morphine in rats in vivo. The SFM model describes a partial (5%-30%) intestinal blood flow perfusing the transporter- and enzyme-rich enterocyte region, whereas the TM describes 100% flow perfusing the intestine as a whole. For the SFM, drugs entering from the circulation are expected to be metabolized to lesser extents by the intestine due to the segregated flow, reflecting the phenomenon of shunting and route-dependent intestinal metabolism. The poor permeability of MG crossing the liver or intestinal basolateral membranes mandates that most of MG that is excreted into bile is hepatically formed, whereas MG that is excreted into urine originates from both intestine and liver metabolism, since MG is effluxed back to blood. The ratio of MG amounts in urine/bile [Formula: see text] for intraduodenal/intravenous dosing is expected to exceed unity for the SFM but approximates unity for the TM. Compartmental analysis of morphine and MG data, without consideration of the permeability of MG and where MG is formed, suggests the ratio to be 1 and failed to describe the kinetics of MG. The observed intraduodenal/intravenous ratio of [Formula: see text] (2.55 at 4 hours) was better predicted by the SFM-PBPK (2.59 at 4 hours) and not the TM-PBPK (1.0), supporting the view that the SFM is superior for the description of intestinal-liver metabolism of morphine to MG. The SFM-PBPK model predicts an appreciable contribution of the intestine to first pass M metabolism.

Physiologically-Based Pharmacokinetic-Pharmacodynamic Modeling of 1α,25-Dihydroxyvitamin D3 in Mice.

1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3] concentrations are regulated by renal CYP27B1 for synthesis and CYP24A1 for degradation. Published plasma and tissue 1,25(OH)2D3 concentrations and mRNA fold change expression of Cyp24a1 and Cyp27b1 following repetitive i.p. injections to C57BL/6 mice (2.5 µg × kg(-1) every 2 days for 4 doses) were fitted with a minimal and full physiologically-based pharmacokinetic-pharmacodynamic models (PBPK-PD). The minimal physiologically-based pharmacokinetic-pharmacodynamic linked model (mPBPK-PD) related Cyp24a1 mRNA fold changes to linear changes in tissue/tissue baseline 1,25(OH)2D3 concentration ratios, whereas the full physiologically-based pharmacokinetic-pharmacodynamic model (PBPK-PD) related measured tissue Cyp24a1 and Cyp27b1 fold changes to tissue 1,25(OH)2D3 concentrations with indirect response, sigmoidal maximal stimulatory effect/maximal inhibitory effect functions. Moreover, the intestinal segregated flow model (SFM) that describes a low and partial intestinal (blood/plasma) flow to enterocytes was nested within both models for comparison with the traditional model for intestine (TM) where the entire flow perfuses the intestine. Both the mPBPK(SFM)-PD and full PBPK(SFM)-PD models described the i.p. plasma and tissue 1,25(OH)2D3 concentrations and fold changes in mRNA expression significantly better than the TM counterparts with F test comparisons. The full PBPK(SFM)-PD fits showed estimates with good precision (lower percentage of coefficient of variation), and the model was more robust in predicting data from escalating i.v. doses (2, 60, and 120 pmol) and the rebound in 1,25(OH)2D3 tissue concentrations after dosing termination. The full PBPK(SFM)-PD model performed the best among the tested models for describing the complex pharmacokinetic-pharmacodynamic interplay among Cyp27b1, Cyp24a1, and 1,25(OH)2D3.