Yoichi Naritomi

Prevention of progressive joint destruction in collagen‐induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR255031

1 FR255031 (2‐[(7S)‐7‐[5‐(4‐ethylphenyl)‐2‐thienyl]‐1,1‐dioxido‐4‐(2‐pyridinylcarbonyl)hexahydro‐1,4‐thiazepin‐7‐yl]‐N‐hydroxyacetamide) is a novel synthetic matrix metalloproteinase (MMP) inhibitor that inhibits human collagenases (MMP‐1, MMP‐8 and MMP‐13), gelatinases (MMP‐2 and MMP‐9) and membrane type 1 MMP (MT1‐MMP/MMP‐14). FR255031 also inhibits rat collagenase and gelatinase. We studied the effect of FR255031 and Trocade, an inhibitor of collagenase and MMP‐14, on a rat collagen‐induced arthritis (CIA) model. 2 Rat CIA was induced by intradermal injection of type II collagen (IIC) and oral administration of FR255031 or Trocade was performed for 28 days. Body weight loss, hind paw swelling, elevation of serum anti‐IIC antibody, and histological and radiographic scores were evaluated. 3 FR255031 markedly inhibited cartilage degradation in a dose‐dependent manner in the CIA model, but Trocade failed to prevent the degradation. 4 FR255031 at a dose of 100 mg kg−1 also had statistically significant effects on bone destruction and pannus formation and on the recovery of body weight loss on day 28. 5 These results indicate that FR255031 is effective for rat CIA, especially on joint cartilage destruction. These data suggest that as well as collagenases or MT‐MMP, gelatinases are also involved in joint destruction in arthritis.

Identification and relative contributions of human cytochrome P450 isoforms involved in the metabolism of glibenclamide and lansoprazole: evaluation of an approach based on the in vitro substrate disappearance rate

1. The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug. 2. Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CLint) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively. 3. CLint of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CLint of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CLint of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4. 4. The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.

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

Abstract 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.

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.

Predictability of plasma concentration–time curves in humans using single-species allometric scaling of chimeric mice with humanized liver

Abstract 1. We used chimeric mice (PXB mice®), which were repopulated with human hepatocytes, to evaluate their predictabilities of human pharmacokinetics. 2. The relationships of total clearance (CLt) and the volume of distribution at steady state (Vdss) between that predicted from single-species allometric scaling (SSS) of PXB mice and the observed human values indicated good correlations for various drugs metabolized by cytochrome P450s (CYPs) and non-CYPs. 3. We examined the Dedrick plot with which the plasma concentration–time curves can exhibit superimposability using SSS of PXB mice for CLt and Vdss. The predicted plasma concentration–time curves using the complex Dedrick plot from PXB mice were generally superimposed with the observed human data. 4. However, the predicted curve of diazepam was not superimposable with the observed profile. Residual mouse hepatocytes in the livers of PXB mice may affect predictability of CLt of diazepam because significant discrepancy of in vitro intrinsic clearance in PXB mouse liver microsomes consisted of low and high replacement of human hepatocytes were observed. 5. The complex Dedrick plot with SSS from PXB mice is useful for predicting the plasma concentration–time curve in drug discovery, although there are some limitations.

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

Abstract 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.

Quantitative Prediction of Human Intestinal Glucuronidation Effects on Intestinal Availability of UDP-Glucuronosyltransferase Substrates Using In Vitro Data

We investigated whether the effects of intestinal glucuronidation on the first-pass effect can be predicted from in vitro data for UDP-glucuronosyltransferase (UGT) substrates. Human in vitro intrinsic glucuronidation clearance (CLint, UGT) for 11 UGT substrates was evaluated using pooled intestinal microsomes (4.00–4620 μl · min−1 · mg−1) and corrected by the free fraction in the microsomal mixture (CLuint, UGT = 5.2–5133 μl · min−1 · mg−1). Eleven UGT substrates were stable against intestinal cytochrome P450, indicating intestinal glucuronidation has a main effect on human intestinal availability. Oral absorbability intestinal availability (FaFg) values were calculated from in vivo pharmacokinetic parameters in the literature (FaFg = 0.01–1.0). It was found that CLuint, UGT and human FaFg have an inverse relationship that can be fitted to a simplified intestinal availability model. Experiments using Supersomes from insect cells expressing UGT isoforms showed that the substrates used were conjugated by various UGT isoforms. These results suggest that combining the simplified intestinal availability model and in vitro conjugation assay make it possible to predict human FaFg regardless of UGT isoform.

Method for predicting human intestinal first-pass metabolism of UGT substrate compounds

As intestinal glucuronidation has been suggested to generate the low oral bioavailability (F) of drugs, estimating its effects would be valuable for selecting drug candidates. Here, we investigated the absorption and intestinal availability (FaFg) in animals, and intrinsic clearance via UDP-glucuronosyltransferase (UGT) in intestinal microsomes (CLint,UGT) for three drug candidates possessing a carboxylic acid group, in an attempt to estimate the impact of intestinal glucuronidation on F and select potential drug candidates with high F in humans. The FaFg values of the three test compounds were low in rats and monkeys (0.16–0.51), and high in dogs (≥0.81). Correspondingly, the CLint,UGT values were high in rats and monkeys (101–731 µL/min/mg), and low in dogs (≤ 59.6 µL/min/mg). A good inverse correlation was observed between FaFg and CLint,UGT, suggesting that intestinal glucuronidation was a major factor influencing FaFg of these compounds. By applying this correlation to FaFg in humans using human CLint,UGT values (26.9–114 µL/min/mg), compounds 1–3 were predicted to have relatively high FaFg. Our approach is expected to be useful for estimating the impact of intestinal glucuronidation on F in animals and semiquantitatively predicting human F for drug candidates.

Discovery of 2-methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}propan-2-ol: a novel, potent and selective type 5 17β-hydroxysteroid dehydrogenase inhibitor.

Type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5), also known as aldo-keto reductase 1C3 (AKR1C3), is a member of the aldo-keto reductase superfamily of enzymes and is expressed in the human prostate. One of the main functions of 17β-HSD5 is to catalyze the conversion of the weak androgen, androstenedione, to the potent androgen, testosterone. The concentration of intraprostatic 5α-dihydrotestosterone (DHT) in patients following chemical or surgical castration has been reported to remain as high as 39% of that of healthy men, with 17β-HSD5 shown to be involved in this androgen synthesis. Inhibition of 17β-HSD5 therefore represents a promising target for the treatment of castration-resistant prostate cancer (CRPC). To investigate this, we conducted high-throughput screening (HTS) and identified compound 2, which displayed a structure distinct from known 17β-HSD5 inhibitors. To optimize the inhibitory activity of compound 2, we first introduced a primary alcohol group. We then converted the primary alcohol group to a tertiary alcohol, which further enhanced the inhibitory activity, improved metabolic stability, and led to the identification of compound 17. Oral administration of compound 17 to castrated nude mice bearing the CWR22R xenograft resulted in the suppression of androstenedione (AD)-induced intratumoral testosterone production. Compound 17 also demonstrated good isoform selectivity, minimal inhibitory activity against either CYP or hERG, and enhanced pharmacokinetic and physicochemical properties.

DEVELOPMENTO F DRUG METABOLISMS TUDY IN DRUG DISCOVERY

It is important to predict and evaluate drug metabolism in drug discovery, because there are many pharmacokinetic problems arising from drug metabolism. Particularly, construction of the rapid and reliable prediction. evaluation methods is important in this study. The drug metabolism items that should be investigated are raised as follows: (1) hepatic clearance, (2) metabolite profile, (3) CYP identification, (4) CYP inhibition, (5) enzyme induction. In this presentation, I will discuss the construction and application of the prediction evaluation methods for hepatic clearance, metabolic profile and CYP inhibition in drug discovery.