Drug Metabolism and Disposition最新文献

Over the past 20 years, quantitative proteomics has contributed a wealth of protein expression data, which are currently used for a variety of systems pharmacology applications, as a complement or a surrogate for activity of the corresponding proteins. A symposium at the 25th North American International Society for the Study of Xenobiotics meeting, in Boston, in September 2023, was held to explore current and emerging applications of quantitative proteomics in translational pharmacology and strategies for improved integration into model-informed drug development based on practical experience of each of the presenters. A summary of the talks and discussions is presented in this perspective alongside future outlook that was outlined for future meetings. SIGNIFICANCE STATEMENT: This perspective explores current and emerging applications of quantitative proteomics in translational pharmacology and precision medicine and outlines the outlook for improved integration into model-informed drug development.

In this study, the nonclinical pharmacokinetics of OLX702A-075-16, an RNA interference therapeutic currently in development, were investigated. OLX702A-075-16 is a novel N-acetylgalactosamine conjugated asymmetric small-interfering RNA (GalNAc-asiRNA) used for the treatment of an undisclosed liver disease. Its unique 16/21-mer asymmetric structure reduces nonspecific off-target effects without compromising efficacy. We investigated the plasma concentration, tissue distribution, metabolism, and renal excretion of OLX702A-075-16 following a subcutaneous administration in mice and rats. For bioanalysis, high-performance liquid chromatography with fluorescence detection was used. The results showed rapid clearance from plasma (0.5 to 1.5 hours of half-life) and predominant distribution to the liver and/or kidney. Less than 1% of the liver concentration of OLX702A-075-16 was detected in the other tissues. Metabolite profiling using liquid chromatography coupled with high-resolution mass spectrometry revealed that the intact duplex OLX702A-075-16 was the major compound in plasma. The GalNAc moiety was predominantly metabolized from the sense strand in the liver, with the unconjugated sense strand of OLX702A-075-16 accounting for more than 95% of the total exposure in the rat liver. Meanwhile, the antisense strand was metabolized by the sequential loss of nucleotides from the 3'-terminus by exonuclease, with the rat liver samples yielding the most diverse truncated forms of metabolites. Urinary excretion over 96 hours was less than 1% of the administered dose in rats. High plasma protein binding of OLX702A-075-16 likely inhibited its clearance through renal filtration. SIGNIFICANCE STATEMENT: This study presents the first comprehensive characterization of the in vivo pharmacokinetics of GalNAc-asiRNA. The pharmacokinetic insights gained from this research will aid in understanding toxicology and efficacy, optimizing delivery platforms, and improving the predictive power of preclinical species data for human applications.

The organic cation transporter (OCT)-1 mediates hepatic uptake of cationic endogenous compounds and xenobiotics. To date, limited information exists on how Oct1/OCT1 functionally develops with age in rat and human livers and how this would affect the pharmacokinetics of OCT substrates in children or juvenile animals. The functional ontogeny of rOct/hOCT was profiled in suspended rat (2-57 days old) and human hepatocytes (pediatric liver tissue donors: age 2-12 months) by determining uptake clearance of 4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide (ASP+) as a known rOct/hOCT probe substrate. mRNA expression was determined in rat liver tissue corresponding to rat ages used in the functional studies, while hOCT1 mRNA expressions were determined in the same hepatocyte batches as those used for uptake studies. Maturation of rOct/hOCT activity and expression were evaluated by comparing values obtained at the various ages to the adult values. Relative to adult values (at 8 weeks), ASP⁺ uptake clearance in suspended rat hepatocytes aged 0, 1, 2, 3, 4, 5, and 6 weeks reached 26%, 29%, 33%, 37%, 72%, 63%, and 71%, respectively. Hepatic Oct1 mRNA expression was consistent with Oct activity (correlation coefficient of 0.92). In human hepatocytes, OCT1 activity was age dependent and also correlated with mRNA levels (correlation coefficient of 0.88). These data show that Oct1/OCT1 activities and expression mature gradually in rat/human liver, thereby mirroring the expression pattern of organic anion transporting polypeptide in rat. These high-resolution transporter ontogeny profiles will allow for more accurate prediction of the pharmacokinetics of OCT1/Oct1 substrates in pediatric populations and juvenile animals. SIGNIFICANCE STATEMENT: Organic cation transporter-1 (OCT1) represents a major drug uptake transporter in human liver. This study provides high-resolution data regarding the age-dependent function of OCT1 in the liver, based on in vitro experiments with rat and human hepatocytes obtained from donors between birth and adulthood. These ontogeny profiles will inform improved age-specific physiologically based pharmacokinetic models for OCT1 drug substrates in neonates, infants, children, and adults.

Sterol 12α-hydroxylase (CYP8B1) is the unique P450 enzyme with sterol 12-oxidation activity, playing an exclusive role in 12α-hydroxylating intermediates along the bile acid (BA) synthesis pathway. Despite the long history of BA metabolism studies, it is unclear whether CYP8B1 catalyzes 12α-hydroxylation of C₂₇ BAs, the key intermediates shuttling between mitochondria and peroxisomes. This work provides robust in vitro evidence that both microsomal and recombinant CYP8B1 enzymes catalyze the 12α-hydroxylation of dihydroxycoprostanic acid (DHCA) into trihydroxycoprostanic acid (THCA). On the one hand, DHCA 12α-hydroxylation reactivity is conservatively detected in liver microsomes of both human and preclinical animals. The reactivity of human tissue fractions conforms well with the selectivity of CYP8B1 mRNA expression, while the contribution of P450 enzymes other than CYP8B1 is excluded by reaction phenotyping in commercial recombinant enzymes. On the other hand, we prepared functional recombinant human CYP8B1 proteins according to a recently published protocol. Titration of the purified CYP8B1 proteins with either C4 (7α-hydroxy-4-cholesten-3-one) or DHCA yields expected blue shifts of the heme Soret peak (type I binding). The recombinant CYP8B1 proteins efficiently catalyze 12α-hydroxylation of both DHCA and C4, with substrate concentration occupying half of the binding sites of 3.0 and 1.9 μM and k_cat of 3.2 and 2.6 minutes^-1, respectively. In summary, the confirmed role of CYP8B1 in 12α-hydroxylation of C₂₇ BAs has furnished the forgotten passageway in the BA synthesis pathway. The present finding might have opened a new window to consider the biology of CYP8B1 in glucolipid metabolism and to evaluate CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases. SIGNIFICANCE STATEMENT: The academic community has spent approximately 90 years interpreting the synthesis of bile acids. However, the 12α-hydroxylation of intermediates catalyzed by CYP8B1 is not completely mapped on the classic pathway, particularly for the C₂₇ bile acids, the pivotal intermediates shuttling between mitochondria and peroxisomes. This work discloses the forgotten 12α-hydroxylation pathway from dihydroxycoprostanic acid into trihydroxycoprostanic acid. The present finding may facilitate evaluating CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases.

Drug-drug interaction (DDI) assessment of therapeutic peptides is an evolving area. The industry generally follows DDI guidelines for small molecules, but the translation of data generated with commonly used in vitro systems to in vivo is sparse. In the current study, we investigated the ability of advanced human hepatocyte in vitro systems, namely HepatoPac, spheroids, and Liver-on-a-chip, to assess potential changes in regulation of CYP1A2, CYP2B6, CYP3A4, SLCO1B1, and ABCC2 in the presence of selected therapeutic peptides, proteins, and small molecules. The peptide NN1177, a glucagon and GLP-1 receptor co-agonist, did not suppress mRNA expression or activity of CYP1A2, CYP2B6, and CYP3A4 in HepatoPac, spheroids, or Liver-on-a-chip; these findings were in contrast to the data obtained in sandwich cultured hepatocytes. No effect of NN1177 on SLCO1B1 and ABCC2 mRNA was observed in any of the complex systems. The induction magnitude differed across the systems (e.g., rifampicin induction of CYP3A4 mRNA ranged from 2.8-fold in spheroids to 81.2-fold in Liver-on-a-chip). Small molecules, obeticholic acid and abemaciclib, showed varying responses in HepatoPac, spheroids, and Liver-on-a-chip, indicating a need for EC₅₀ determinations to fully assess translatability data. HepatoPac, the most extensively investigated in this study (3 donors), showed high potential to investigate DDIs associated with CYP regulation by therapeutic peptides. Spheroids and Liver-on-a-chip were only assessed in one hepatocyte donor and further evaluations are required to confirm their potential. This study establishes an excellent foundation toward the establishment of more clinically-relevant in vitro tools for evaluation of potential DDIs with therapeutic peptides. SIGNIFICANT STATEMENT: At present, there are no guidelines for drug-drug interaction (DDI) assessment of therapeutic peptides. Existing in vitro methods recommended for assessing small molecule DDIs do not appear to translate well for peptide drugs, complicating drug development for these moieties. Here, we establish evidence that complex cellular systems have potential to be used as more clinically-relevant tools for the in vitro DDI evaluation of therapeutic peptides.

Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called adverse analytical finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offenses and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from nonathletes were analyzed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e., samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the nonapproved proliferator-activated receptor delta agonist GW1516) in a total of 81 samples, with 91% of these suspected cases being verified by the confirmation method. In addition to the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. SIGNIFICANCE STATEMENT: This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing.

A STING (stimulator of interferon genes) agonist GSK3996915 under investigation in early discovery for hepatitis B was orally dosed to a mouse model for understanding the parent drug distribution in liver, the target organ. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was used to quantify the distribution of GSK3996915 in liver collected from mice administered a single oral dose at 90 mg/kg. GSK3996915 was detected with a zonal distribution localized in the portal triad and highly concentrated in the main bile ducts, indicating clearance through biliary excretion. High spatial resolution imaging showed the distribution of the parent drug localized to the cellular populations in the sinusoids, including the Kupffer cells. Additionally, a series of drug-related metabolites were observed to be localized in the central zones of the liver. These results exemplify the potential of utilizing MALDI IMS for measuring not only quantitative drug distribution and target exposure but also drug metabolism and elimination in a single suite of experiments. SIGNIFICANCE STATEMENT: An integrated imaging approach utilizing matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) complemented with immunohistochemistry (IHC) and histology was used to address the question of target exposure at the cellular level. Localized quantification of the parent drug in the target organ and identification of potential metabolites in the context of tissue histology were also achieved in one experimental suite to support characterization of pharmacokinetic properties of the drug in the early discovery stage.

The regulation of drug-metabolizing enzymes and transporters by cytokines has been extensively studied in vitro and in clinic. Cytokine-mediated suppression of cytochrome P450 (CYP) or drug transporters may increase or decrease the systemic clearance of drug substrates that are primarily cleared via these pathways; neutralization of cytokines by therapeutic proteins may thereby alter systemic exposures of such drug substrates. The Food and Drug Administration recommends evaluating such clinical drug interactions during clinical development and has provided labeling recommendations for therapeutic proteins. To determine the clinical relevance of these drug interactions to dose adjustments, trends in steady-state exposures of CYP-sensitive substrates coadministered with cytokine modulators as reported in the University of Washington Drug Interaction Database were extracted and examined for each of the CYPs. Coadministration of cytochrome P450 family 3 subfamily A (CYP3A) (midazolam/simvastatin), cytochrome P450 subfamily 2C19 (omeprazole), or cytochrome P450 subfamily 1A2 (caffeine/tizanidine) substrates with anti-interleukin-6 and with anti-interleukin-23 therapeutics led to changes in systemic exposures of CYP substrates ranging from ∼ -58% to ∼35%; no significant trends were observed for cytochrome P450 subfamily 2D6 (dextromethorphan) and cytochrome P450 subfamily 2C9 (warfarin) substrates. Although none of these changes in systemic exposures have been reported as clinically meaningful, dose adjustment of midazolam for optimal sedation in acute care settings has been reported. Simulated concentration-time profiles of midazolam under conditions of elevated cytokine levels when coadministered with tocilizumab, suggest a ∼six- to sevenfold increase in midazolam clearance, suggesting potential implications of cytokine-CYP drug interactions on dose adjustments of sensitive CYP3A substrates in acute care settings. Additionally, this article also provides a brief overview of nonclinical and clinical assessments of cytokine-CYP drug interactions in drug discovery and development. SIGNIFICANCE STATEMENT: There has been significant progress in understanding cytokine-mediated drug interactions for CYP-sensitive substrates. This article provides an overview of the progress in this field, including a trend analysis of systemic exposures of CYP-sensitive substrates coadministered with anti-interleukin therapeutics. In addition, the review also provides a perspective of current methods used to assess these drug interactions during drug development and a focus on individualized medicine, particularly in acute care settings.

Human organic anion transporting polypeptide (OATP) 1B1 and 1B3 are two highly homologous liver-specific uptake transporters. However, 2',7'-dichlorofluorescein (DCF) is preferably transported by OATP1B1. In the present study, the molecular mechanisms for the selective transport of DCF by OATP1B1 were investigated by constructing and characterizing an array of OATP1B1/1B3 chimeras and site-directed mutagenesis. Our results show that transmembrane domain (TM) 10 is crucial for the surface expression and function of OATP1B1, in which Q541 and L545 play the most important roles in DCF transport. Replacement of TM10 in OATP1B1 with its OATP1B3 counterpart led to OATP1B1's complete intracellular retention. Q541 and L545 may interact with DCF directly via hydrogen bonding and hydrophobic interactions. The decrease of DCF uptake by Q541A and L545S was due to their reduced binding affinity for DCF as compared with OATP1B1. In addition, Q541 and L545 are also crucial for the transport of estradiol-17β-glucuronide (E17βG) but not for the transport of estrone-3-sulfate (E3S), indicating different interaction modes between DCF/E17βG and E3S in OATP1B1. Taken together, Q541 and L545 in TM10 are critical for OATP1B1-mediated DCF uptake, but their effect is substrate-dependent. SIGNIFICANCE STATEMENT: The key TMs and amino acid residues for the selective transport of DCF by OATP1B1 were identified. TM10 is crucial for the surface expression and function of OATP1B1. Within TM10, Q541 and L545 played the most significant roles and affected the function of OATP1B1 in a substrate-dependent manner. This information is crucial for a better understanding of the mechanism of the multispecificity of OATP1B1 and as a consequence the mechanism of OATP1B1-mediated drug-drug interactions.