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RO7119929 is being developed as an orally administered prodrug of the TLR7-specific agonist and active drug, RO7117418, for the treatment of patients with solid tumours.In this publication, we present a case study wherein the human pharmacokinetics and pharmacological active dose were prospectively predicted following oral administration of the prodrug.A simple translational pharmacokinetic-pharmacodynamic strategy was applied to predict the pharmacological active dose of the prodrug in human. In vivo studies in monkey showed that an unbound plasma exposure of active drug of 1.5 ng/mL elicited secretion of key serum pharmacodynamic cytokine and chemokine biomarkers in monkey. This threshold of 1.5 ng/mL was close to the minimum effective concentration of active drug required to induce cytokine secretion in human peripheral blood mononuclear cells (3 ng/mL).Measured in vitro physicochemical and biochemical properties of the prodrug and active drug were applied as input parameters in physiologically based pharmacokinetic models to predict the pharmacokinetics of active drug after oral dosing of the prodrug in humans. Then, using the PBPK model, a dose which delivered an unbound plasma Cmax in line with the target pharmacodynamic threshold of 1.5 ng/mL was found. This defined the lowest pharmacologically active dose as 3 mg.The prodrug entered the clinic in 2020 in patients with primary or secondary liver cancers. Clear pharmacodynamic, transient, and dose-dependent cytokine induction was observed at prodrug doses > 1 mg.

Brexpiprazole, a serotonin-dopamine activity modulator, is indicated for the treatment of schizophrenia and also adjunctive therapy to antidepressants for the treatment of Major Depressive Disorder. To determine the drug-drug interaction risk for cytochrome P450, and SLC and ABC transporters, brexpiprazole and its metabolite, DM-3411 were assessed in this in vitro investigation.Brexpiprazole exhibited weak inhibitory effects (IC 50 >13 μmol/L) on CYP2C9, CYP2C19, CYP2D6 and CYP3A4 activities, but had moderate inhibitor activity on CYP2B6 (IC 50 8.19 μmol/L). The ratio of systemic unbound concentration (3.8 nmol/L) to the K i value was sufficiently low. DM-3411 had comparable inhibitory potentials with brexpiprazole only for CYP2D6 and CYP3A4. The mRNA expressions of CYP1A2, CYP2B6 and CYP3A4 were not changed by the exposure of brexpiprazole to human hepatocytes.Brexpiprazole and DM-3411 exhibited weak or no inhibitory effects for hepatic and renal transporters (OATPs, OATs, OCTs, MATE1, and BSEP), except for MATE-2K (0.156 μmol/L of DM-3411), even for which the ratio to systemic unbound concentration (5.3 nmol/L) was sufficiently low.Brexpiprazole effected the functions of P-gp and BCRP with IC 50 values of 6.31 and 1.16 μmol/L, respectively, however, the pharmacokinetic alteration was not observed in the clinical concomitant study on P-gp and BCRP substrates.These in vitro data suggest that brexpiprazole is unlikely to cause clinically relevant drug interactions resulting from the effects on CYPs or transporters mediating the absorption, metabolism, and/or disposition of co-administered drugs.

1. AFQ056 phenotyping results indicate that CYP1A1 is responsible for the formation of the oxidative metabolite, M3. In line with the predominant assumption that CYP1A1 is mainly expressed in extrahepatic tissues, only traces of M3 were detected in hepatic systems. The aim of this study was to investigate the pulmonary CYP1A1 mediated metabolism of AFQ056 in rat. 2. Western blot analysis confirmed that CYP1A1 is expressed in rat lung albeit at low levels. M3 formation was clearly observed in recombinant rat CYP1A1, lung microsomes and lung tissue slices and was strongly inhibited by ketoconazole in the incubations. As CYP3A4 and CYP2C9 metabolites were only observed at trace levels, we concluded that the reduced M3 formation was due to CYP1A1 inhibition. 3. AFQ056 lung clearance (CL lung ) as estimated from in vitro data was predicted to be negligible (<1% pulmonary blood flow). This was confirmed by in vivo experiments where intravenous and intra-arterial dosing to rats failed to show significant pulmonary extraction. 4. While rat lung may make a contribution to the formation of M3, it is unlikely to be the only organ involved in this process and further experiments are required to investigate the potential metabolic elimination routes for AFQ056.

1. Esterases may play a major role in the clearance of drugs with functional groups amenable to hydrolysis, particularly in the case of ester prodrugs. To understand the processes involved in the elimination of such drugs, it is necessary to determine the esterases involved. However, the tools currently available for this enzyme phenotyping are relatively scarce. 2. The work was aimed at summarizing the selectivity of esterase inhibitors for carboxylesterases 1 and 2 (CES1 and CES2) in the human liver to clarify their suitability for esterase phenotyping. Eserine, at around 10 μM, was found to be a highly specific CES2 inhibitor, whereas other esterase inhibitors turned out less selective. When used together with tacrine, which inhibits cholinesterases but not CES, and ethylenediaminetetraacetic acid (inhibitor of paraoxonases), the involvement of the hydrolyzing esterases in the hepatic clearance of a drug can be elucidated. 3. The second approach to esterase phenotyping is based on data from recombinant or isolated esterases, together with relative activity factors, which relate their activities to those of the same enzymes in subcellular fractions. 4. These two approaches will help to characterize the hydrolytic metabolism of drug candidates in a similar manner as practiced routinely for the oxidative metabolism by cytochrome P450 enzymes.

1. Chlorzoxazone (CLZ) is currently being used as a marker substrate in vitro/vivo studies to quantify cytochrome P450 2E1 (CYP2E1) activity in humans. Although in CLZ 6-hydroxylation several CYPs are responsible, previous studies have presented the monophasicity of the reaction in human liver microsomes (HLMs). Furthermore, the Km values of CYP2E1 for the 6-hydroxylation in HLMs were reported to be lower than those of its recombinant enzymes. 2. This study aimed to provide the comprehensive Km values for the CLZ 6-hydroxylation in HLMs using CYP antibodies. The Eadie-Hofstee plots revealed a biphasic profile and indicate that the reaction was mainly mediated by CYP1A2 as well as CYP2E1. The formation of 6-hydroxychlorzoxazone was more specific for CYP2E1 activity at higher substrate concentration in HLMs. 3. Moreover, KOH as a vehicle for substrate or sucrose included in HLMs preparation had some effect on the activity of CLZ 6-hydroxylase. These constituents seemed to be casually related to the apparent monophasic kinetics and variability in Km values for the CLZ 6-hydroxylation in HLMs. 4. The Km of CYP1A2 and CYP2E1 in HLMs was 3.8 µmol/L and 410 µmol/L, respectively, and the value of CYP2E1 was close to that of recombinant CYP2E1.

Cynomolgus monkeys are widely used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage. Characterization of cynomolgus monkey P450s such as the mRNA expression level, substrate specificity, and inhibitor selectivity were conducted to provide helpful information in designing monkey in vivo studies and monkey-to-human extrapolation. The expression levels of 12 monkey P450 mRNAs, which are considered to be important P450 subfamilies in drug metabolism, were investigated in the liver, small intestine (duodenum, jejunum, and ileum), and colon of individual monkeys. 3. In vitro activities and intrinsic clearance values were determined in monkey intestinal and liver microsomes (MIM and MLM, respectively) using nine typical oxidative reactions for human P450s. Paclitaxel 6α-hydroxylation, diclofenac 4′-hydroxylation, and S-mephenytoin 4′-hydroxylation showed low activities in MIM and MLM. IC₅₀ values of eight selective inhibitors of human P450s were determined in MIM and MLM. Inhibitory effects of furafylline and sulfaphenazole were weak in monkeys on phenacetin O-deethylation and diclofenac 4′-hydroxylation, respectively. These results show profiles of monkey P450s in both the intestine and liver in detail and contribute to a better understanding of the species difference in substrate specificity and inhibitor selectivity between cynomolgus monkeys and humans.

In this study the tissue distribution of radioactivity in pregnant and lactating rats was investigated by quantitatively determining radioactivity concentrations and by whole-body autoradioluminograms after a single oral administration of 14C-YM758. In addition, the transfer of radioactivity into the reproductive tissues, foetus, and milk is discussed in terms of the localization of transporters in syncytiotrophoblast and mammary gland. The radioactivity concentrations in the liver were the highest of all the tissues and organs tested at all the sampling times. The radioactivity in main tissues (liver and kidney), including reproductive tissues (amniotic fluid, placenta, ovary, and uterus), was not retained for a long time, as in the plasma. The tissue/plasma (T/P) ratio of radioactivity in the foetus was below 1.0, which might be due to Mdr1-mediated export of YM758 into blood via the blood-placenta barrier since YM758 is a substrate for hMDR1, not for hBCRP/rBcrp. The T/P ratio of radioactivity in the maternal milk 1 and 4 h after oral administration of 14C-YM758 was 7.2 and 11.0, respectively. To understand better the distribution of new drugs into the reproductive tissues/milk, and to interpret further the results of reproductive safety studies for drug development, the contribution of transporters expressed in the blood-placenta barrier and mammary gland to the drug-transfer into placenta and milk should be considered.

The inhibitory effects of cationic drugs (beta-adrenoreceptor antagonists, calcium (Ca)-channel blocker, I(f) channel inhibitor, antiarrhythmic drugs, and antibacterial drugs) that inhibit 1-methyl-4-phenylpyridinium (MPP) and/or metformin uptake into hOCT1-3/rOct1-3-expressing cells and human/rat hepatocytes were investigated in this study. The drug-drug interaction (DDI) potential of these drugs for the hOCT/rOct-mediated hepatic/renal uptake process was also assessed. The IC(50) values of cardiovascular drugs, including an I(f) channel inhibitor with a new mechanism of action, were greater for hOCT2/rOct2 than those for hOCT1/rOct1 or hOCT3/rOct3. No species differences in these values were observed between hOCTs and rOcts. As for hOCT2-mediated uptake, the IC(50) values of quinidine and the I(f) channel inhibitor for metformin uptake were lower than those for MPP uptake. However, previous clinical studies found that the IC(50) values of these drugs for hOCT1/rOct1 and hOCT2/rOct2 were much greater than their unbound plasma concentrations, which suggests that the DDIs of these cationic compounds may not be related to hOCT/rOct-mediated hepatic/renal uptake pathways. In addition, investigation of the luminal transporters of cationic compounds in the kidney, as well as the in vitro DDI potential of their inhibitors, is important for the clarification of cationic compound DDIs in humans.

1. YM758 is a novel If channel inhibitor for the treatment of stable angina and atrial fibrillation. The absorption, distribution, and excretion of YM758 have been investigated in albino and non-albino rats after a single oral administration of (14)C-YM758 monophosphate. 2. YM758 was well absorbed from all segments of the gastrointestinal tract except for the stomach. After oral administration, the ratio of AUC(0-1 h) between the plasma concentrations of radioactivity and the unchanged drug was estimated to be 17.7%, which suggests metabolism. 3. The distribution of the radioactivity derived from (14)C-YM758 in tissues was evaluated both in albino and non-albino rats. The radioactivity concentrations in most tissues were higher than those in plasma, which indicates that the radioactivity is well distributed to tissues. Extensive accumulation and slower elimination of radioactivity were noted in the thoracic aorta of albino and non-albino rats as well as in the eyeballs of non-albino rats. The recovery rates of radioactivity in urine and bile after oral dosing to bile duct-cannulated albino rats were 17.8% and 57.3%, respectively. 4. These results suggest that YM758 was extensively absorbed, subjected to metabolism, and excreted mainly into the bile after oral administration to rats, and extensive accumulation of the unchanged drug and/or metabolites into tissues such as the thoracic aorta and eyeballs was observed.

The contribution of organic cation transporters to the saturable component in the hepatic uptake of 1-methyl-4-phenylpyridinium (MPP), tetraethylammonium (TEA), cimetidine, and metformin was examined by the use of human/rat organic cation transporter (hOCT1/rOct1)-expressing cells and human/rat hepatocytes. Transfection of rOct1 resulted in a considerable increase in the uptake of metformin, whereas that of hOCT1 resulted in only a slight increase. All test compounds (MPP, TEA, cimetidine, and metformin) accumulated in human and rat hepatocytes in a carrier-mediated manner. The Km values for the uptake of MPP, TEA, cimetidine, and metformin into human and rat hepatocytes were comparable with those into hOCT1 and rOct1-expressing cells, respectively. In addition, the relative uptake activities, which were obtained by normalizing the intrinsic uptake clearances of TEA, cimetidine, and metformin against those values of MPP in human and rat hepatocytes, were similar with the uptake activities in hOCT1 and rOct1, respectively. These results suggest that the saturable component in the hepatic uptake of these cationic compounds may be mediated mainly by hOCT1/rOct1; therefore, it is meaningful to evaluate the saturable uptake profile of cationic compounds by the liver using both hOCT1/rOct1-expressing cells and human/rat hepatocytes.

In this study, the comparison of the transport of substrates (1-methyl-4-phenylpydinium (MPP) and tetraethyl ammonium (TEA)) and the inhibition potency of the inhibitors (biguanides and H(2)-blockers) for human and rat organic cation transporters (hOCTs and rOcts), and the inhibition type of inhibitors for these transporters were investigated using HEK293 cells that stably express hOCT/rOct. The concentration-dependent uptake of [(3)H]-MPP and [(14)C]-TEA by hOCT1-3/rOct1-3 had K(m) values similar to those in the literature. It was also deduced that MPP and TEA are competitive inhibitors for hOCT1-2/rOct1-2. The K(i) values for phenformin inhibition of [(3)H]-MPP and [(14)C]-TEA uptake by hOCT1-3/rOct1-3 were lower than that for metformin. The [(3)H]-MPP uptake by hOCT1/rOct1 and hOCT3/rOct3 was inhibited by famotidine and ranitidine whereas that by hOCT2/rOct2 was not. The inhibitory potency of cimetidine for hOCT1-2 was very weak. In most cases, the differences in the V(max)/K(m) values of substrates and the K(i) values of inhibitors between hOCT and rOct were minor. The acquisition of information on OCT/Oct mediated-transport and/or inhibition such as that presented in this report is very useful for further understanding of certain aspects of uptake, distribution, and excretion for drug candidates.

1. The oxidative one-carbon cleavage reaction in the octyl side chain of olanexidine [1-(3,4-dichlorobenzyl)-5-octylbiguanide], a new potent biguanide antiseptic, was characterized in dog liver microsomes. 2. Olanexidine was initially biotransformed to a monohydroxylated metabolite, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-2-octanol (DM-215), and DM-215 was subsequently oxidized to the diol derivative, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-1,2-octandiol (DM-220). DM-220 was further biotransformed to 2-hydroxy aldehyde derivative, 2-hydroxy carboxylic acid derivative, and an oxidative C-1-C-2 bond cleavage metabolite, 7-[5-(3,4-dichlorobenzyl)-1-biguanidino] heptanoic acid [DM-223 (C7), a seven-carbon chain derivative], after incubation with dog liver microsomes. 3. DM-223 formation required NADPH as a cofactor and was inhibited by quinidine and quinine, relatively selective inhibitors of CYP2D subfamilies in dogs. 4. The results suggest that the one-carbon fragment of the octyl side chain of olanexidine could be removed by the oxidative C-C bond cleavage with the possible involvement of cytochrome P450 systems such as CYP2D subfamily. This oxidative C-C bond cleavage reaction by cytochrome P450s could play an important role in the removal of one-carbon fragment of other drugs or endogenous compounds containing aliphatic chains.

1. The biotransformation of 1-(3,4-dichlorobenzyl)-5-octylbiguanide (OPB-2045), a new potent biguanide antiseptic, was investigated in male beagle dogs. Urinary and faecal excretion of unchanged compound and metabolites were studied following a single subcutaneous injection of 14C-labeled compound at a dose of 1 mg/kg. 2. Four urinary metabolites were structually identified using synthetic standards and/or spectral data as 3,4-dichlorobenzoic acid, 6-[5-(3,4-dichlorobenzyl)-1-biguanidino] hexanoic acid (DM-210), 4-[5-(3,4-dichlorobenzl)-1-biguanidino] butanoic acid (DM-212) and 5-[5-(3,4-dichlorobenzyl)-1-biguanidino] pentanoic acid (DM-213). 3. The predominant radioactive substances in the excreta were DM-213 and DM-210 at 26.1% and 25.5%, respectively, of the dose. No unchanged compound was detected in the urine, and in the faeces it was only 2% of the dose.

1. The metabolism of carteolol, a beta-adrenoceptor blocking drug, was investigated in male Sprague-Dawley rat liver microsomes. 2. The formation of 8-hydroxycarteolol was the principal metabolic pathway of carteolol in vitro and followed Michaelis-Menten kinetics with a K(m) = 11.0 +/- 5.4 microM and a Vmax = 1.58 +/- 0.64 nmol/min/nmol P450 respectively (mean +/- SD, n = 5). Eadie-Hofstee plot analysis of carteolol 8-hydroxylase activity confirmed single-enzyme Michaelis-Menten kinetics. 3. The cytochrome P450 isoforms involved in 8-hydroxylation of carteolol were investigated using selective chemical inhibitors and polyclonal anti-P450 antibodies. Quinine (Ki = 0.06 microM) and quinidine (Ki = 2.0 microM), selective inhibitors of CYP2D1, competitively inhibited 8-hydroxycarteolol formation. Furthermore, only anti-human CYP2D6 antibody inhibited this reaction. 4. These results suggest that carteolol is metabolized to 8-hydroxycarteolol by CYP2D1. The K(m) of carteolol for CYP2D1 in male rat liver microsomes was much greater than those of propranolol or bunitrolol, indicating that carteolol has a lower affinity for CYP2D1 compared with these other beta-adrenoceptor blocking drugs.