Drug clearance by aldehyde oxidase: can we avoid clinical failure?

Despite increased awareness of aldehyde oxidase (AO) as a major drug-metabolising enzyme, predicting the pharmacokinetics of its substrates remains challenging. Several drug candidates have been terminated due to high clearance, which were subsequently discovered to be AO substrates. Even retrospective extrapolation of human clearance, from models more sensitive to AO activity, often resulted in underprediction.The questions of the current work thus were: Is there an acceptable degree of in vitro AO metabolism that does not result in high in vivo human clearance? And, if so, how can this be predicted?We built an in vitro/in vivo correlation using known AO substrates, combining multiple in vitro parameters to calculate the blood metabolic clearance mediated by AO (CLbAO). This value was compared with observed blood clearance (CLb-obs), establishing cut-off CLbAO values, to discriminate between low and high CLb-obs. The model was validated using additional literature compounds, and CLb-obs was predicted in the correct category.This simple, categorical, semi-quantitative yet multi-factorial model is readily applicable in drug discovery. Further, it is valuable for high-clearance compounds, as it predicts the CLb group, rather than an exact CLb value, for the substrates of this poorly-characterised enzyme.

Characterization of Metabolites of α-mangostin in Bio-samples from SD Rats by UHPLC-Q-exactive Orbitrap MS.

BACKGROUND: α-mangostin, a typical xanthone, often exists in Garcinia mangostana L. (Clusiaceae). α-mangostin was found to have a wide range of pharmacological properties. However, its specific metabolic route in vivo remains unclear, while these metabolites may accumulate to exert pharmacological effects, too. OBJECTIVE: This study aimed to clarify the metabolic pathways of α-mangostin after oral administration to the rats. METHODS: Here, an UHPLC-Q-Exactive Orbitrap MS was used for the detection of potential metabolites formed in vivo. A new strategy for the identification of unknown metabolites based on typical fragmentation routes was implemented. RESULTS: A total of 42 metabolites were detected, and their structures were tentatively identified in this study. The results showed that major in vivo metabolic pathways of α-mangostin in rats included methylation, demethylation, methoxylation, hydrogenation, dehydrogenation, hydroxylation, dehydroxylation, glucuronidation, and sulfation. CONCLUSIONS: This study is significant to expand our knowledge of the in vivo metabolism of α-mangostin and to understand the mechanism of action of α-mangostin in rats in vivo.

Spreadsheet-Based Minimal Physiological Models for the Prediction of Clearance of Therapeutic Proteins in Pediatric Patients.

There is a growing interest in the use of physiologically based pharmacokinetic (PBPK) models as clinical pharmacology drug development tools. In PBPK modeling, not every organ or physiological parameter is required, leading to the development of a minimal PBPK (mPBPK) model, which is simple and efficient. The objective of this study was to streamline mPBPK modeling approaches and enable straightforward prediction of clearance of protein-based products in children. Four mPBPK models for scaling clearance from adult to children were developed and evaluated on Excel spreadsheets using (1) liver and kidneys; (2) liver, kidneys, and skin; (3) liver, kidneys, skin, and lymph; and (4) interstitial, lymph, and plasma volume. There were 35 therapeutic proteins with a total of 113 observations across different age groups (premature neonates to adolescents). For monoclonal and polyclonal antibodies, more than 90% of observations were within a 0.5- to 2-fold prediction error for all 4 methods. For nonantibodies, 79% to 100% of observations were within the 0.5- to 2-fold prediction error for the 4 different methods. Methods 1 and 4 provided the best results, >90% of the total observations were within the 0.5- to 2-fold prediction error for all 3 classes of protein-based products across a wide age range. The precision of clearance prediction was comparatively lower in children ≤2 years of age vs older children (>2 years of age) with methods 1 and 4 predicting 80% to 100% and 75% to 90% of observations within the 0.5- to 2-fold prediction error, respectively. The results of the study indicated that mPBPK models can be developed on spreadsheets, with acceptable performance for prediction of clearance.

Metabolism and Mass Balance of the Novel Nonsteroidal Androgen Receptor Inhibitor Darolutamide in Humans.

The biotransformation and excretion of darolutamide were investigated in a phase I study. Six healthy male volunteers received a single dose of 300 mg 14C-darolutamide as an oral solution in the fasted state. Plasma, urine, and feces samples were analyzed for mass balance evaluation by liquid scintillation counting (LSC). Metabolite profiling and identification were determined using liquid chromatography mass-spectrometry with off-line radioactivity detection using LSC. Complete mass balance was achieved, with mean radioactivity recovery of 95.9% within 168 hours (63.4% in urine, 32.4% in feces). The administered 1:1 ratio of (S,R)- and (S,S)-darolutamide changed to approximately 1:5, respectively, in plasma. Darolutamide and the oxidation product, keto-darolutamide, were the only components quantifiable by LSC in plasma, accounting for 87.4% of total radioactivity, with a 2.1-fold higher plasma exposure for keto-darolutamide. Aside from darolutamide, the most prominent metabolites in urine were O-glucoronide (M-7a/b) and N-glucuronide (M-15a/b), as well as pyrazole sulfates (M-29, M-24) and glucuronides (M-21, M-22) resulting from oxidative cleavage of the parent. The darolutamide diastereomers were mainly detected in feces. In vitro assays showed that darolutamide metabolism involves a complex interplay between oxidation and reduction, as well as glucuronidation. Interconversion of the diastereomers involves oxidation to keto-darolutamide, primarily mediated by CYP3A4, followed by reduction predominantly catalyzed by cytosolic reductase(s), with aldo-keto reductase 1C3 playing the major role. The latter reaction showed stereoselectivity with preferential formation of (S,S)-darolutamide. SIGNIFICANCE STATEMENT: The metabolism and excretion of darolutamide in humans revealed that oxidation (CYP3A4) and glucuronidation (UGT1A9, UGT1A1) were the main metabolic routes of elimination. Direct excretion also contributed to overall clearance. The two pharmacologically equipotent diastereomers of darolutamide interconvert primarily via oxidation to the active metabolite keto-darolutamide, followed by reduction predominantly by cytosolic reductase(s). The latter reaction showed stereoselectivity with preferential formation of (S,S)-darolutamide. Data indicate a low drug-drug interaction potential of darolutamide with inducers or inhibitors of metabolizing enzymes.

Absorption, Metabolism, Distribution, and Excretion of Letermovir.

BACKGROUND: Letermovir is approved for prophylaxis of cytomegalovirus infection and disease in cytomegalovirus-seropositive hematopoietic stem-cell transplant (HSCT) recipients. OBJECTIVE: HSCT recipients are required to take many drugs concomitantly. The pharmacokinetics, absorption, distribution, metabolism, and excretion of letermovir and its potential to inhibit metabolizing enzymes and transporters in vitro were investigated to inform on the potential for drug-drug interactions (DDIs). METHODS: A combination of in vitro and in vivo studies described the absorption, distribution, metabolism, and routes of elimination of letermovir, as well as the enzymes and transporters involved in these processes. The effect of letermovir to inhibit and induce metabolizing enzymes and transporters was evaluated in vitro and its victim and perpetrator DDI potentials were predicted by applying the regulatory guidance for DDI assessment. RESULTS: Letermovir was a substrate of CYP3A4/5 and UGT1A1/3 in vitro. Letermovir showed concentration- dependent uptake into organic anionic transporting polypeptide (OATP)1B1/3-transfected cells and was a substrate of P-glycoprotein (P-gp). In a human ADME study, letermovir was primarily recovered as unchanged drug and minor amounts of a direct glucuronide in feces. Based on the metabolic pathway profiling of letermovir, there were few oxidative metabolites in human matrix. Letermovir inhibited CYP2B6, CYP2C8, CYP3A, and UGT1A1 in vitro, and induced CYP3A4 and CYP2B6 in hepatocytes. Letermovir also inhibited OATP1B1/3, OATP2B1, OAT3, OCT2, BCRP, BSEP, and P-gp. CONCLUSION: The body of work presented in this manuscript informed on the potential for DDIs when letermovir is administered both intravenously and orally in HSCT recipients.

In Silico Pharmacokinetic Study of Vancomycin Using PBPK Modeling and Therapeutic Drug Monitoring.

BACKGROUND: Vancomycin has been in clinical use for nearly 50 years and remains the first-line treatment option for Gram-positive infections, including methicillin-resistant Staphylococcus aureus (MRSA). There are multiple strategies to monitor therapy and adjust the dose of this antibiotic. AUC24/MIC ratio has been demonstrated to be the best parameter to predict the effectiveness and safety of vancomycin, and a target ratio of ≥400 is recommended. Still, trough and peak serum levels at steady-state conditions have been used in clinical settings as an accurate and practical method to monitor vancomycin. METHODS: In this work, we collected and analyzed clinical information of patients being treated in a hospital center in Porto (Portugal) and studied the pharmacokinetics of vancomycin in silico, developing several physiologically based pharmacokinetic (PBPK) models using simulation software GastroPlus™. Different dosages and treatment regimens were studied, and the influence of patients' age, weight and renal function was evaluated; a simulation population was also performed. RESULTS: A linear effect of dose and a significant influence of weight and renal function in plasmatic levels of vancomycin was observed. CONCLUSION: The results of this work corroborate the accumulation of vancomycin in plasma and identify some parameters that influence the pharmacokinetics of this antibiotic. The importance of therapeutic monitoring of vancomycin is highlighted, and the usefulness of in silico tools, namely PBPK modeling, is demonstrated.

In Vivo Gene Expression Profile of Human Intestinal Epithelial Cells: From the Viewpoint of Drug Metabolism and Pharmacokinetics.

Orally administered drugs are absorbed and metabolized in the intestine. To accurately predict pharmacokinetics in the intestine, it is essential to understand the intestinal expression profiles of the genes related to drug absorption, distribution, metabolism, and excretion (ADME). However, in many previous studies, gene expression analysis in the intestine has been carried out using specimens from patients with cancer. In this study, to obtain more accurate gene expression profiles, biopsy samples were collected under endoscopic observation from the noninflammatory regions of 14 patients with inflammatory bowel disease, and RNA-seq analysis was performed. Gene expression analysis of drug-metabolizing enzymes (cytochromes P450), non-cytochrome P450 enzymes, nuclear receptors, drug-conjugating enzymes (UDP-glucuronosyltransferases and sulfotransferases), and apical and basolateral drug transporters was performed in biopsy samples from the duodenum, ileum, colon, and rectum. The proportions of the cytochromes P450 expressed in the ileum were 25% (CYP3A4), 19% (CYP2C18), and 14% (CYP3A5). CYP3A4 and CYP2C19 were highly expressed in the duodenum and ileum, but not in the colon and rectum. In the ileum, apical transporters such as P-gp, peptide transporter 1, breast cancer resistance protein, MRP2, and ASBT were strongly expressed, and the expression levels of P-gp and ASBT in the ileum were higher than those in other regions. In the ileum, basolateral transporters such as OSTα, OSTß, and MRP3 were strongly expressed. We succeeded in obtaining gene expression profiles of ADME-related genes in human intestinal epithelial cells in vivo. We expect that this information would be useful for accurate prediction of the pharmacokinetics of oral drugs. SIGNIFICANCE STATEMENT: To obtain gene expression profiles of ADME-related genes in human intestinal epithelial cells in vivo, biopsy samples were collected under endoscopic observation from the noninflammatory regions of 14 patients with inflammatory bowel disease, and RNA-seq analysis was performed. Gene expression profiles of drug-metabolizing enzymes (cytochromes P450), non-cytochrome P450 enzymes, nuclear receptors, drug-conjugating enzymes (UDP-glucuronosyltransferases and sulfotransferases), and apical and basolateral drug transporters in biopsy samples from the duodenum, ileum, colon, and rectum were obtained in this study.

Impact of Plasma Protein Binding in Drug Clearance Prediction: A Data Base Analysis of Published Studies and Implications for In Vitro-In Vivo Extrapolation.

Plasma protein-mediated uptake (PMU) and its effect on clearance (CL) prediction have been studied in various formats; however, a comprehensive analysis of the overall impact of PMU on CL parameters from hepatocyte assays (routinely used for IVIVE) has not previously been performed. The following work collated data reflecting the effect of PMU for 26 compounds with a wide variety of physicochemical, drug, and in vivo CL properties. PMU enhanced the unbound intrinsic clearance in vitro (CLint,u in vitro) beyond that conventionally calculated using fraction unbound and was correlated with the unbound fraction of drug in vitro and in plasma (fup) and absolute unbound intrinsic clearance in vivo (CLint,u in vivo) in both rat and human hepatocytes. PMU appeared to be more important for highly bound (fup < 0.1) and high CLint,u in vivo drugs. These trends were independent of species, assay conditions, ionization, and extended clearance classification system group, although the type of plasma protein used in in vitro assays may require further investigation. Such generalized trends (spanning fup 0.0008-0.99) may suggest a generic mechanism behind PMU; however, multiple drug-dependent mechanisms are also possible. Using the identified relationship between the impact of PMU on CLint,u in vitro and fup, PMU-enhanced predictions of CLint,u in vivo were calculated for both transporter substrates and metabolically cleared drugs. PMU was accurately predicted, and incorporation of predicted PMU improved the IVIVE of hepatic CL, with an average fold error of 1.17 and >50% of compounds predicted within a 2-fold error for both rat and human data sets (n ≥ 100). SIGNIFICANCE STATEMENT: Current strategies for prediction of hepatic clearance from in vitro data are recognized to be inaccurate, but they do not account for PMU. The impact of PMU on CLint,u in vitro is wide ranging and can be predicted based on fraction unbound in plasma and applied to CLint,u in vitro values obtained by standard procedures in the absence of plasma protein. Such PMU-enhanced predictions improved IVIVE, and future studies may easily incorporate this PMU relationship to provide more accurate IVIVE.

Prediction of Transporter-Mediated Drug-Drug Interactions and Phenotyping of Hepatobiliary Transporters Involved in the Clearance of E7766, a Novel Macrocycle-Bridged Dinucleotide.

E7766 represents a novel class of macrocycle-bridged dinucleotides and is under clinical development for immuno-oncology. In this report, we identified mechanism of systemic clearance E7766 and investigated the hepatobiliary transporters involved in the disposition of E7766 and potential drug interactions of E7766 as a victim of organic anion-transporting polypeptide (OATP) inhibitors. In bile-duct cannulated rats and dogs, E7766 was mainly excreted unchanged in bile (>80%) and to a lesser extent in urine (<20%). Sandwich-cultured human hepatocytes (SCHHs), transfected cells, and vesicles were used to phenotype the hepatobiliary transporters involved in the clearance of E7766. SCHH data showed temperature-dependent uptake of E7766 followed by active biliary secretion. In vitro transport assays using transfected cells and membrane vesicles confirmed that E7766 was a substrate of OATP1B1, OATP1B3, and multidrug resistance-associated protein 2. Phenotyping studies suggested predominant contribution of OATP1B3 over OATP1B1 in the hepatic uptake of E7766. Studies in OATP1B1/1B3 humanized mice showed that plasma exposure of E7766 increased 4.5-fold when coadministered with Rifampicin. Physiologically based pharmacokinetic models built upon two independent bottom-up approaches predicted elevation of E7766 plasma exposure when administered with Rifampicin, a clinical OATP inhibitor. In conclusion, we demonstrate that OATP-mediated hepatic uptake is the major contributor to the clearance of E7766, and inhibition of OATP1B may increase its systemic exposure. Predominant contribution of OATP1B3 in the hepatic uptake of E7766 was observed, suggesting polymorphisms in OATP1B1 would be unlikely to cause variability in the exposure of E7766. SIGNIFICANCE STATEMENT: Understanding the clearance mechanisms of new chemical entities is critical to predicting human pharmacokinetics and drug interactions. A physiologically based pharmacokinetic model that incorporated parameters from mechanistic in vitro and in vivo experiments was used to predict pharmacokinetics and drug interactions of E7766, a novel dinucleotide drug. The findings highlighted here may shed a light on the pharmacokinetic profile and transporter-mediated drug interaction propensity of other dinucleotide drugs.

Creatinine-Based Renal Function Assessment in Pediatric Drug Development: An Analysis Using Clinical Data for Renally Eliminated Drugs.

The estimated glomerular filtration rate (eGFR) equations based on serum creatinine (SCR) have been used for pediatric dose adjustment in drug labeling. This study evaluated the performance of those equations in estimating individual clearance of drugs that are predominantly eliminated by glomerular filtration, using clinical data from the renally eliminated drugs gadobutrol, gadoterate, amikacin, and vancomycin. The eGFR was compared with the observed drug clearance (CL) in 352 pediatric patients from birth to 12 years of age. Multiple eGFR equations overestimated the drug CL on average, including the original and bedside Schwartz equations, which showed an average eGFR/CL ratio between 1 and 3. Further analysis with bedside Schwartz equation showed a higher eGFR/CL ratio in the subjects with a lower SCR or CL. Supraphysiological eGFR as high as 380 mL/min/1.73 m2 was obtained using the bedside Schwartz equation for some of the subjects, most of whom are children < 2 years of age with SCR < 0.2 mg/dL. Excluding the subjects with supraphysiological eGFR from the analysis did not change the overall trend of overestimation. In conclusion, Schwartz equations led to an overestimation of drug clearance for the drugs evaluated. When greater precision is required in predicting eGFR for pediatric patients, such as in drug dosing, revised k constants for the Schwartz equation or new methods of glomerular filtration rate estimation may be necessary.

The role of antibiotic pharmacokinetic studies performed post-licensing.

Post-licensing pharmacometric studies can provide a better understanding of the pharmacokinetic (PK) alterations in special patient populations and may lead to better clinical outcomes. Some patient populations exhibit markedly different pathophysiology to general ward patients or healthy individuals. This may be developmental (paediatric patients), a manifestation of an underlying disease pathology (patients with obesity or haematological malignancies) or due to medical interventions (critically ill patients receiving extracorporeal therapies). This paper outlines the factors that affect the PK of special patient populations and describes some novel methods of antimicrobial administration that may increase antimicrobial concentrations at the site of infection and improve treatment of severe infection.

A plausible way for excretion of metal nanoparticles via active targeting.

Even so, the metal nanoparticles (metal NPs) have attractive optical and biomedical applications, the translation of metal NPs into the clinical practice remains a challenge due to their severe accumulation in the body. Active targeting to renal podocytes opens the door for enhancing kidney targeting and clearance. The goal of this study was to assess the excretion of larger particle size through kidney podocyte via active targeting. To reach this goal, PEGylated quantum dots (QDs) were coated with vapreotide (VAP) for selectively reaching somatostatin receptors (SSTRs) expressed in the podocyte cells. This QDs-VAP was tested on isolated primary podocytes, while the flow cytometry (FACS), confocal microscopy (CLSM), and inductively coupled plasma mass spectrometry (ICP-MS) were used to confirm this hypothesis. The results showed highly specific interactions with podocyte cells as detected by FACS, and CLSM. Moreover, ICP-MS demonstrated higher amount of QDs in the podocyte cells one-hour post-incubation (67.99% ID/g tissue), while the unmodified QDs did not accumulate. This study confirmed that QDs-VAP can target the podocyte's SSTRs then can be cleared via podocyte cells. Moreover, these results are considered as a highly promising approach for future therapy, targeting, clearance, and diagnosis of podocyte-associated diseases.

Drugs in pregnancy: Pharmacologic and physiologic changes that affect clinical care.

Pharmacologic interventions play a major role in obstetrical care throughout pregnancy, labor and delivery and the postpartum. Traditionally, obstetrical providers have utilized standard dosing regimens developed for non-obstetrical indications based on pharmacokinetic knowledge from studies in men or non-pregnant women. With the recognition of pregnancy as a special pharmacokinetic population in the late 1990s, investigators have begun to study drug disposition in this unique patient dyad. Many of the basic physiologic changes that occur during pregnancy have significant impact on drug absorption, distribution and clearance. Activity of Phase I and Phase II drug metabolizing enzymes are differentially altered by pregnancy, resulting in drug concentrations sufficiently different for some medications that efficacy or toxicity is affected. Placental transporters play a major dynamic role in determining fetal drug exposure. In the past two decades, we have begun to expand our understanding of obstetrical pharmacology; however, to truly optimize pharmacologic care of our pregnant patients and their developing fetus, additional research is critically needed.

Pharmacokinetic studies in pregnancy.

The effects of the many biochemical and physiologic changes of pregnancy on the dose-response relationship of drugs administered to pregnant women are poorly understood. The dose-response relationship is affected by pharmacokinetics, or what the body does to a drug, and pharmacodynamics, or what a drug does to the body. Insights into the potential effects of the changes of pregnancy on one aspect of the dose-response relationship of a drug can be obtained by studying the pharmacokinetics of the drug in the various stages of pregnancy and the postpartum period. There are several available approaches to studying pharmacokinetic changes in pregnancy. Single trough screening studies can provide qualitative estimates of elimination clearance, which with the dosing rate determines the steady-state drug concentration, throughout pregnancy and into the postpartum period. Population pharmacokinetic studies such as two stage pharmacokinetic studies and studies using a nonlinear mixed effects pharmacokinetic modeling approach can characterize pharmacokinetic changes more rigorously.

Population Pharmacokinetics of Amikacin Administered Once Daily in Patients with Different Renal Functions.

The aim of this work was to evaluate the pharmacokinetics of amikacin in Mexican patients with different renal functions receiving once-daily dosing regimens and the influence of clinical and demographical covariates that may influence the optimization of this antibiotic. A prospective study was performed in a total of 63 patients with at least one determination of amikacin plasma concentration. Population pharmacokinetic (PK) parameters were estimated by nonlinear mixed-effects modeling; validations were performed for dosing recommendation purposes based on PK/pharmacodynamic simulations. The concentration-versus-time data were best described by a one-compartment open model with proportional interindividual variability associated with amikacin clearance (CL) and volume of distribution (V); residual error followed a homoscedastic trend. Creatinine clearance (CLCR) and ideal body weight (IBW) demonstrated significant influence on amikacin CL and V, respectively. The final model [CL (liters/h) = 7.1 × (CLCR/130)0.84 and V (liters) = 20.3 × (IBW/68)2.9] showed a mean prediction error of 0.11 mg/liter (95% confidence interval, -3.34, 3.55) in the validation performed in a different group of patients with similar characteristics. There is a wide variability in amikacin PK parameters in Mexican patients. This leads to inadequate dosing regimens, especially in patients with augmented renal clearance (CLCR of >130 ml/min). Optimization based on the final population PK model in Mexican patients may be useful, since reliability and clinical applicability have been demonstrated in this study.

In Vivo-to-In Vitro Extrapolation of Transporter-Mediated Renal Clearance: Relative Expression Factor Versus Relative Activity Factor Approach.

About 30% of approved drugs are cleared predominantly by renal clearance (CLr). Of these, many are secreted by transporters. For these drugs, in vitro-to-in vivo extrapolation of transporter-mediated renal secretory clearance (CLsec,plasma) is important to prospectively predict their renal clearance and to assess the impact of drug-drug interactions and pharmacogenetics on their pharmacokinetics. Here we compared the ability of the relative expression factor (REF) and the relative activity factor (RAF) approaches to quantitatively predict the in vivo CLsec,plasma of 26 organic anion transporter (OAT) substrates assuming that OAT-mediated uptake is the rate-determining step in the CLsec,plasma of the drugs. The REF approach requires protein quantification of each transporter in the tissue (e.g., kidney) and transporter-expressing cells, whereas the RAF approach requires the use of a transporter-selective probe substrate (both in vitro and in vivo) for each transporter of interest. For the REF approach, 50% and 69% of the CLsec,plasma predictions were within 2- and 3-fold of the observed values, respectively; the corresponding values for the RAF approach were 65% and 81%. We found no significant difference between the two approaches in their predictive capability (as measured by accuracy and bias) of the CLsec,plasma or CLr of OAT drugs. We recommend that the REF and RAF approaches can be used interchangeably to predict OAT-mediated CLsec,plasma Further research is warranted to evaluate the ability of the REF or RAF approach to predict CLsec,plasma of drugs when uptake is not the rate-determining step. SIGNIFICANCE STATEMENT: This is the first direct comparison of the relative expression factor (REF) and relative activity factor (RAF) approaches to predict transporter-mediated renal clearance (CLr). The RAF, but not REF, approach requires transporter-selective probes and that the basolateral uptake is the rate-determining step in the CLr of drugs. Given that there is no difference in predictive capability of the REF and RAF approach for organic anion transporter-mediated CLr, the REF approach should be explored further to assess its ability to predict CLr when basolateral uptake is not the sole rate-determining step.