[An introduction to QSP modeling for pharmacologists].

Quantitative systems pharmacology (QSP) is an emerging field of modeling technologies that describes the dynamic interaction between biological systems and drugs. Recently, QSP is increasingly being applied to pharmaceutical drug discovery and development, and used for various types of decision makings. In contrast to empirical and statistical models, QSP represents complex systems of human physiology by integrating comprehensive biological information, hence, it can address various purposes including target and/or disease-related biomarker identification, hypothesis testing, and prediction of clinical efficacy or toxicity. On the other hand, structures of QSP models become quite complicated with huge amount of biological components, therefore, close collaboration between pharmacologists having profound knowledge of biology and drug metabolism and pharmacokinetics (DMPK) scientists, experts of model building, is crucial for QSP development and implementation. This article introduces, from DMPK scientists to pharmacologists, main features of QSP and its applications in pharmaceutical industries, and discusses challenges and future perspectives for effective utilization in drug discovery and development.

Application of a physiologically based pharmacokinetic model for the prediction of mirabegron plasma concentrations in a population with severe renal impairment.

We previously verified a physiologically based pharmacokinetic (PBPK) model for mirabegron in healthy subjects using the Simcyp Simulator by incorporating data on the inhibitory effect on cytochrome P450 (CYP) 2D6 and a multi-elimination pathway mediated by CYP3A4, uridine 5'-diphosphate-glucuronosyltransferase (UGT) 2B7 and butyrylcholinesterase (BChE). The aim of this study was to use this PBPK model to assess the magnitude of drug-drug interactions (DDIs) in an elderly population with severe renal impairment (sRI), which has not been evaluated in clinical trials. We first determined the system parameters, and meta-analyses of literature data suggested that the abundance of UGT2B7 and the BChE activity in an elderly population with sRI was almost equivalent to and 20% lower than that in healthy young subjects, respectively. Other parameters, such as the CYP3A4 abundance, for an sRI population were used according to those built into the Simcyp Simulator. Second, we confirmed that the PBPK model reproduced the plasma concentration-time profile for mirabegron in an sRI population (simulated area under the plasma concentration-time curve (AUC) was within 1.5-times that of the observed value). Finally, we applied the PBPK model to simulate DDIs in an sRI population. The PBPK model predicted that the AUC for mirabegron with itraconazole (a CYP3A4 inhibitor) was 4.12-times that in healthy elderly subjects administered mirabegron alone, and predicted that the proportional change in AUC for desipramine (a CYP2D6 substrate) with mirabegron was greater than that in healthy subjects. In conclusion, the PBPK model was verified for the purpose of DDI assessment in an elderly population with sRI.

Possible involvement of the µ opioid receptor in the antinociception induced by sinomenine on formalin-induced nociceptive behavior in mice.

Sinomenine, an alkaloid originally isolated from the roots and the rhizome of Sinomenium acutum is used as a traditional Chinese herbal medicines for rheumatoid arthritis and neuralgia. The aims of this study were to investigate the effects of oral administration of shinomenine on formalin-induced nociceptive behavior in mice and the opioid receptor subtypes involved in the antinociceptive effects of sinomenine. Our findings showed that a single dose of oral-administrated sinomenine inhibited the formalin induced licking and biting responses in a dose-dependent manner. Intraperitoneal pretreatment with naloxone hydrochloride, an opioid receptor antagonist, and ß-funaltrexamine hydrochloride (ß-FNA), a selective µ-opioid receptor antagonist, significantly attenuated sinomenine induced antinociception, but not by naltrindole, a nonselective δ-opioid receptor antagonist and nor-binaltorphimine, a selective κ-opioid receptor antagonist. Furthermore, in western blot analysis, oral administration of sinomenine resulted in a significant blockage of spinal extracellular signal-regulated protein kinase (ERK1/2) activation induced by formalin. Naloxone hydrochloride and ß-FNA significantly reversed the blockage of spinal ERK1/2 activation induced by sinomenine. These results suggest that sinomenine-induced anti nociceptive effect and blockage of spinal ERK1/2 activation may be triggered by activation of µ-opioid receptors.

Physiologically-based pharmacokinetic modeling for mirabegron: a multi-elimination pathway mediated by cytochrome P450 3A4, uridine 5'-diphosphate-glucuronosyltransferase 2B7, and butyrylcholinesterase.

This was the first study to construct a physiologically-based pharmacokinetic (PBPK) model for mirabegron which incorporates the overall elimination pathways of metabolism by cytochrome P450 (CYP) 3A4, uridine 5'-diphosphate-glucuronosyltransferase (UGT) 2B7, and butyrylcholinesterase (BChE) and renal excretion. The objective was to assess the risk of drug-drug interactions (DDIs) by estimating the contribution of each elimination pathway and simulating the magnitude of the DDIs with UGT2B7 inhibitors. A PBPK model for mirabegron was constructed to reproduce the plasma concentration-time curves from a phase 1 study and the magnitude of the DDI with ketoconazole taking into account the overall elimination pathways. The PBPK model was subsequently verified using data from other DDI studies. The constructed PBPK model estimated the contribution for each elimination pathway: 44% and 29% for CYP3A4 and UGT2B7 in the liver, 1.6% for UGT2B7 in the kidney, 3.2% for BChE in plasma, and 22% for renal excretion. Co-administration of probenecid (an UGT2B7 inhibitor) or fluconazole (an UGT2B7 and CYP3A4 inhibitor) was predicted to increase area under the curve for mirabegron to 115% or 174%, respectively. In conclusion, PBPK modeling and simulation revealed a low DDI risk for mirabegron following co-administration with BChE or UGT2B7 inhibitors.

The Impact of Endogenous Breast Cancer Resistance Protein on Human P-Glycoprotein-Mediated Transport Assays Using LLC-PK1 Cells Transfected With Human P-Glycoprotein.

Lilly Laboratories cell porcine kidney 1 (LLC-PK1) cells transfected with human P-glycoprotein (LLC-PK1-P-gp) are widely used in transport assays to identify drug candidates that function as substrates of this efflux transporter. Endogenous transporters expressed in LLC-PK1 cells may complicate the interpretation of findings from P-gp-mediated transport assays. We investigated the impact of porcine breast cancer resistance protein (Bcrp) in P-gp-mediated transport assays in LLC-PK1 cells. Porcine Bcrp mRNA was detected in both LLC-PK1 wildtype (WT) and LLC-PK1-P-gp cells by quantitative RT-PCR. To investigate the activity and impact of porcine Bcrp, we conducted transport assays using 6 typical BCRP substrates in LLC-PK1 cells. Efflux ratios (ER) of the 6 BCRP substrates in LLC-PK1 WT cells were >2, and were reduced in the presence of the BCRP inhibitor Ko143. The efflux activities of the 6 BCRP substrates were confirmed using MDCKII cells transfected with human BCRP. Net ERs of prazosin and fluvastatin, dual substrates of P-gp and BCRP, determined by dividing ERs in LLC-PK1-P-gp cells by those in LLC-PK1 WT cells, were <2, but increased to >2 in the presence of Ko143. These results indicated that endogenous Bcrp in LLC-PK1 cells was involved in the transport of BCRP substrates and may interfere with the identification of P-gp substrates.

Transcriptome analysis of a Pseudomonas aeruginosasn-glycerol-3-phosphate dehydrogenase mutant reveals a disruption in bioenergetics.

Pseudomonas aeruginosa causes acute and chronic human infections and is the major cause of morbidity and mortality in cystic fibrosis (CF) patients. We previously determined that the sn-glycerol-3-phosphate dehydrogenase encoded by glpD plays a larger role in P. aeruginosa physiology beyond its role in glycerol metabolism. To better understand the effect of a glpD mutation on P. aeruginosa physiology we compared the transcriptomes of P. aeruginosa strain PAO1 and the PAO1ΔglpD mutant using RNA-seq analysis. We determined that a null mutation of glpD significantly altered amino acid metabolism in P. aeruginosa and affected the production of intermediates that are channelled into the tricarboxylic acid cycle. Moreover, the loss of glpD induced a general stress response mediated by RpoS in P. aeruginosa. Several other phenotypes observed for the P. aeruginosa glpD mutant include increased persister cell formation, reduced extracellular ATP accumulation and increased heat output. Taken together, these findings implicate sn-glycerol-3-phosphate dehydrogenase as a key player in energy metabolism in P. aeruginosa.

Quantitative LC/ESI-SRM/MS of antibody biopharmaceuticals: use of a homologous antibody as an internal standard and three-step method development.

Monoclonal antibody-based therapeutic agents (antibody drugs) have attracted considerable attention as a new type of drug. Concomitantly, the use of quantitative approaches for characterizing antibody drugs, such as liquid chromatography (LC)-mass spectrometry (MS), has increased. Generally, selective quantification of antibody drugs is done using unique peptides from variable regions (V H and V L) as surrogate peptides. Further, numerous internal standards (ISs) such as stable isotope-labeled (SIL)-intact proteins and SIL-surrogate peptides are used. However, developing LC-MS methodology for characterizing antibody drugs is time-consuming and costly. Therefore, LC-MS is difficult to apply for this purpose, particularly during the drug discovery stage when numerous candidates must be evaluated. Here, we demonstrate an efficient approach to developing a quantitative LC/electrospray ionization (ESI)-selected reaction monitoring (SRM)/MS method for characterizing antibody drugs. The approach consists of the following features: (i) standard peptides or SIL-IS are not required; (ii) a peptide from the homologous monoclonal antibody serves as an IS; (iii) method development is monitored using a spiked plasma sample and one quantitative MS analysis; and (iv) three predicted SRM assays are performed to optimize quantitative SRM conditions such as transition, collision energy, and declustering potential values. Using this strategy, we developed quantitative SRM methods for infliximab, alemtuzumab, and bevacizumab with sufficient precision (<20%)/accuracy (<±20%) for use in the drug discovery stage. We have also demonstrated that choosing a higher homologous peptide pair (from analyte mAb/IS mAb) is necessary to obtain the sufficient precision and accuracy. Graphical abstract ᅟ.

Recent Progress in Hepatocyte Culture Models and Their Application to the Assessment of Drug Metabolism, Transport, and Toxicity in Drug Discovery: The Value of Tissue Engineering for the Successful Development of a Microphysiological System.

Tissue engineering technology has provided many useful culture models. This article reviews the merits of this technology in a hepatocyte culture system and describes the applications of the sandwich-cultured hepatocyte model in drug discovery. In addition, we also review recent investigations of the utility of the 3-dimensional bioprinted human liver tissue model and spheroid model. Finally, we present the future direction and developmental challenges of a hepatocyte culture model for the successful establishment of a microphysiological system, represented as an organ-on-a-chip and even as a human-on-a-chip. A merit of advanced culture models is their potential use for detecting hepatotoxicity through repeated exposure to chemicals as they allow long-term culture while maintaining hepatocyte functionality. As a future direction, such advanced hepatocyte culture systems can be connected to other tissue models for evaluating tissue-to-tissue interaction beyond cell-to-cell interaction. This combination of culture models could represent parts of the human body in a microphysiological system.

High resolution imaging of intracellular oxygen concentration by phosphorescence lifetime.

Optical methods using phosphorescence quenching by oxygen are suitable for sequential monitoring and non-invasive measurements for oxygen concentration (OC) imaging within cells. Phosphorescence intensity measurement is widely used with phosphorescent dyes. These dyes are ubiquitously but heterogeneously distributed inside the whole cell. The distribution of phosphorescent dye is a major disadvantage in phosphorescence intensity measurement. We established OC imaging system for a single cell using phosphorescence lifetime and a laser scanning confocal microscope. This system had improved spatial resolution and reduced the measurement time with the high repetition rate of the laser. By the combination of ubiquitously distributed phosphorescent dye with this lifetime imaging microscope, we can visualize the OC inside the whole cell and spheroid. This system uses reversible phosphorescence quenching by oxygen, so it can measure successive OC changes from normoxia to anoxia. Lower regions of OC inside the cell colocalized with mitochondria. The time-dependent OC change in an insulin-producing cell line MIN6 by the glucose stimulation was successfully visualized. Assessing the detailed distribution and dynamics of OC inside cells achieved by the presented system will be useful to understanding a physiological and pathological oxygen metabolism.

Development and validation of semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-glycoprotein for high-throughput screening.

Transport assays using P-gp-expressing cell lines are commonly used to identify P-gp substrates and inhibitors in drug discovery. The P-gp cell-based assay is performed manually in 12- or 24-well plates and requires improvement for high-throughput screening. In this study, we established an efficient semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-gp. The protocol was optimized with a microplate washer for exchanging media and buffer to enhance throughput. P-gp substrates and inhibitors, and the paracellular marker Dextran Texas Red® were used to validate the 96-well transport assay. Cell monolayer integrity after washing by a microplate washer was confirmed by measuring paracellular permeability of Dextran Texas Red. Permeability and net flux ratio of the P-gp substrates and the inhibitory potency of the P-gp inhibitors were comparable in 24- and 96-well plates. The regression value of net flux ratio of P-gp substrates was high between the two formats (r²=0.99). The optimized 96-well transport assay using the microplate washer was found to be an efficient high-throughput screening tool that provided the same quality data as the 24-well plate for the identification of P-gp substrates and inhibitors in drug discovery.

Prediction of hepatic and intestinal glucuronidation using in vitro-in vivo extrapolation.

The accurate prediction of hepatic (Fh) and intestinal availability (Fg) is vital for determining human pharmacokinetics. To predict these PK parameters for cytochrome P450 (P450) metabolism, in vitro-in vivo extrapolation (IVIVE) using hepatic microsomes, hepatocytes, and intestinal microsomes has been actively investigated. However, IVIVE has not been sufficiently evaluated for non-P450 enzymes. UDP-glucuronosyltransferase (UGT) is a non-P450 enzyme that catalyzes glucuronidation, a major pathway for drugs possessing carboxylic acid, hydroxyl, and amine moieties. In drug metabolism, UGT is the most important enzyme after P450, and prediction of Fh for UGT substrates has mainly been attempted using hepatic models based on the clearance concepts. While various approaches for achieving improved prediction of clearance have been investigated--such as the addition of bovine serum albumin to microsomal incubation mixtures--optimized in vitro methods that utilize both hepatic microsomes and hepatocytes for more accurate prediction are still required. Although application of the simplified intestinal availability (SIA) model is effective in predicting the Fg of UGT substrates, this model is limited to compounds with high oral absorption. In this review, we discuss the current state, issues, and future directions of predicting Fh and Fg for glucuronidation.

Production of xylitol and tetrahydrofurfuryl alcohol from xylan in napier grass by a hydrothermal process with phosphorus oxoacids followed by aqueous phase hydrogenation.

The production of xylitol and tetrahydrofurfuryl alcohol (THFA) from napier grass was studied using two steps: a hydrothermal process with phosphorus oxoacids followed by aqueous phase hydrogenation with Pd/C. Xylose obtained from the napier grass by the hydrothermal treatment with 3.0 wt% phosphorous acid was subsequently converted into xylitol at 51.6% yield of the xylan in napier grass by hydrogenation with 5.0 wt% Pd/C. The furfural produced from napier grass with a 3.0 wt% phosphoric acid treatment was also directly subjected to the hydrogenation as a hydrolysate to yield 41.4% THFA based on the xylan in napier grass. The yields of xylitol and THFA obtained by hydrogenation using the napier grass hydrolysate containing xylose or furfural were almost the same as those of hydrogenation using commercial materials. To our knowledge, this is the first report on the production of THFA in high yield by hydrogenation directly from biomass hydrolysate.

Species differences in intestinal glucuronidation activities between humans, rats, dogs and monkeys.

1. Glucuronidation via UDP-glucuronosyltransferase (UGT) in the intestine has been reported to influence the pharmacokinetics (PK) of drugs; however, information concerning the differences in activity between species is limited. Here, we investigated the in vitro and in vivo activities of intestinal glucuronidation for 17 UGT substrates in humans, rats, dogs and monkeys. 2. Although in vitro intrinsic clearance (CLint,u,UGT) in intestinal microsomes showed a good correlation between humans and laboratory animals, values tended to be lower in humans than in laboratory animals. The ratio of CLint,u,UGT in the absence and presence of bovine serum albumin differed between species. In vivo, the fraction of drug absorbed (FaFg) in humans correlated with that in dogs and monkeys, but not in rats. 3. While an inverse correlation between CLint,u,UGT and FaFg was observed in each species, the CLint,u,UGT values in the intestinal microsomes corresponding to FaFg values in dogs were three to four times higher than in other animals. 4. These results indicate the need for a degree of caution when extrapolating PK data from laboratory animals to humans.

Comparison of intestinal metabolism of CYP3A substrates between rats and humans: application of portal-systemic concentration difference method.

1. Rats are frequently used in pharmacokinetic studies during drug discovery. However, there is limited information regarding species differences in intestinal availability (Fg) between rats and humans. 2. Here, we directly estimated the fraction of dose absorbed in the portal vein (FaFg) of rats for nine CYP3A substrates using portal-systemic concentration difference method and compared them with human FaFg. No distinct difference in FaFg between the two species was observed, and seven of the nine compounds were within a two-fold difference. Given that their net fraction of dose absorbed (Fa) are expected to be high, this result indicates a moderate correlation in Fg between the two species. 3. In contrast, the in vitro intrinsic clearance (CLint,u) in rat intestinal microsomes tended to be lower than that in humans, and the correlation between intestinal CLint,u and FaFg in rats was poor compared with that in humans. 4. Our finding indicates that rats are appropriate animals for evaluation of the intestinal absorption and metabolism of CYP3A substrates. However, a degree of caution is required when estimating rat Fg from rat intestinal microsomes due to the low metabolic activity and the poor correlation between in vitro and in vivo intestinal metabolism.

Production of monosaccharides from napier grass by hydrothermal process with phosphoric acid.

The production of monosaccharides from napier grass was investigated in the presence of acid catalysts using the hydrothermal process. When the napier grass was treated with 3 wt.% phosphoric acid at 160°C for 15min, the xylose yield reached 10.3 wt.%, corresponding to 72.0% of the xylan in it, whereas glucose was hardly obtained. A combined process was then conducted using an 85 wt.% phosphoric acid treatment at 60 °C for 1h followed by a hydrothermal treatment with 3 wt.% phosphoric acid. In the initial treatment with concentrated phosphoric acid the most of xylan was hydrolyzed to xylose, and the crystalline cellulose was converted to its amorphous form. The hydrolysis of cellulose to glucose was significantly enhanced during the following hydrothermal process with 3 wt.% phosphoric acid at 200 °C for 8 min. Consequently, 77.2% yield of xylose and 50.0% yield of glucose were obtained from the combined process.

Measurement of soil bacterial colony temperatures and isolation of a high heat-producing bacterium.

BACKGROUND: The cellular temperatures of microorganisms are considered to be the same as those of their surroundings because the cellular volume is too small to maintain a cellular temperature that is different from the ambient temperature. However, by forming a colony or a biofilm, microorganisms may be able to maintain a cellular temperature that is different from the ambient temperature. In this study, we measured the temperatures of bacterial colonies isolated from soils using an infrared imager and investigated the thermogenesis by a bacterium that increases its colony temperature. RESULTS: The temperatures of some colonies were higher or lower than that of the surrounding medium. A bacterial isolate with the highest colony temperature was identified as Pseudomonas putida. This bacterial isolate had an increased colony temperature when it grew at a temperature suboptimal for its growth. Measurements of heat production using a microcalorimeter showed that the temperature of this extraordinary, microcalorimetrically determined thermogenesis corresponded with the thermographically observed increase in bacterial colony temperature. When investigating the effects of the energy source on this thermal behavior, we found that heat production by this bacterium increased without additional biomass production at a temperature suboptimal for its growth. CONCLUSIONS: We found that heat production by bacteria affected the bacterial colony temperature and that a bacterium identified as Pseudomonas putida could maintain a cellular temperature different from the ambient temperature, particularly at a sub-optimal growth temperature. The bacterial isolate P. putida KT1401 increased its colony temperature by an energy-spilling reaction when the incubation temperature limited its growth.

Synergistic antitumor activities of sepantronium bromide (YM155), a survivin suppressant, in combination with microtubule-targeting agents in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) has a poor prognosis compared to other subtypes, and effective treatment options are limited to cytotoxic agents, including microtubule-targeting agents, due to the lack of molecular targets. Here, we examined the combined effect of sepantronium bromide (YM155) and microtubule-targeting agents in TNBC models. The combination of YM155 with docetaxel showed synergistic antiproliferative and caspase 3/7-inducing effects in MRK-nu-1 and MDA-MB-453 human TNBC cell lines in vitro. YM155 also synergistically enhanced the efficacies of other microtubule-targeting agents, including paclitaxel and vinorelbine, which induced accumulation of survivin at the G2/M phase, whereas it did not affect the efficacy of doxorubicin. Combination treatment with YM155 and microtubule-targeting agents decreased the accumulation of survivin at the G2/M phase and induced greater apoptosis than either single agent alone. Further, combination treatment with YM155 and docetaxel also had a synergistic antitumor effect, achieving complete regression without exacerbation of body weight loss in all mice, in a MRK-nu-1 human TNBC xenograft model. These results suggest that survivin inhibition synergistically sensitize human TNBC cells to microtubule-targeting agents.

Improvement of tumor localization of photosensitizers for photodynamic therapy and its application for tumor diagnosis.

Photodynamic therapy (PDT) and photodynamic diagnosis of cancer are widely used in clinical fields. These are performed using photosensitizers. Many metalloporphyrin-related compounds have been developed as photosensitizers for use in PDT, and these tumor localization ability have been improved in recent research. Moreover, the precursor of porphyrin 5-aminolevulinic acid is used in fluorescence diagnosis using its tumor localization ability. In this review, these applications of photosensitizers in cancer therapy and diagnosis are summarized.

Porphyrins in urine after administration of 5-aminolevulinic acid as a potential tumor marker.

BACKGROUND: Tumor markers are commonly used for cancer screening and as indicators of therapeutic effects. Certain types of tumor have been known to produce a variety of porphyrins after 5-aminolevulinic acid (ALA) administration. In this study, porphyrins in tumor-bearing mouse urine were analyzed after oral administration of ALA in order to identify new tumor markers excreted in the urine. METHODS: Porphyrin concentrations in the urine of tumor-bearing mice were measured after administration of 1.0mg of ALA (approximately 50mgkg(-1)). RESULTS: Porphyrin concentrations in the urine of tumor-bearing mice increased after administration of ALA. HPLC analysis of the urine revealed the existence of uroporphyrin (UP) and coproporphyrin (CP) in the urine of ALA-treated tumor-bearing mice. Furthermore, at 3h after ALA administration, UP concentrations in the urine of tumor-bearing mice significantly increased compared to those in the urine of normal mice. CONCLUSION: These results suggest that UP as a precursor of heme detected in the urine of tumor-bearing mice after ALA administration is a potential marker of tumor development.