Synthesis and structure activity relationships of carbamimidoylcarbamate derivatives as novel vascular adhesion protein-1 inhibitors.

Vascular adhesion protein-1 (VAP-1) is a promising therapeutic target for the treatment of diabetic nephropathy. Here, we conducted structural optimization of the glycine amide derivative 1, which we previously reported as a novel VAP-1 inhibitor, to improve stability in dog and monkey plasma, and aqueous solubility. By chemical modification of the right part in the glycine amide derivative, we identified the carbamimidoylcarbamate derivative 20c, which showed stability in dog and monkey plasma while maintaining VAP-1 inhibitory activity. We also found that conversion of the pyrimidine ring in 20c into saturated rings was effective for improving aqueous solubility. This led to the identification of 28a and 35 as moderate VAP-1 inhibitors with excellent aqueous solubility. Further optimization led to the identification of 2-fluoro-3-{3-[(6-methylpyridin-3-yl)oxy]azetidin-1-yl}benzyl carbamimidoylcarbamate (40b), which showed similar human VAP-1 inhibitory activity to 1 with improved aqueous solubility. 40b showed more potent ex vivo efficacy than 1, with rat plasma VAP-1 inhibitory activity of 92% at 1h after oral administration at 0.3mg/kg. In our pharmacokinetic study, 40b showed good oral bioavailability in rats, dogs, and monkeys, which may be due to its improved stability in dog and monkey plasma.

Synthesis and pharmacological evaluation of glycine amide derivatives as novel vascular adhesion protein-1 inhibitors without CYP3A4 and CYP2C19 inhibition.

Vascular adhesion protein-1 (VAP-1) is a promising therapeutic target for the treatment of diabetic nephropathy. Here, we conducted optimization studies of our lead compound 1, which we previously reported as a novel VAP-1 inhibitor, to enhance the inhibition of human VAP-1 and to reduce CYP3A4 and CYP2C19 inhibition. As a result, we identified 3-chloro-4-{4-[5-(3-{[glycyl(methyl)amino]methyl}phenyl)pyrimidin-2-yl]piperazin-1-yl}benzoic acid (17h) as a novel orally active VAP-1 inhibitor, with 14-fold increased human VAP-1 inhibitory activity compared to 1, without CYP3A4 and CYP2C19 inhibition. Oral administration of 17h significantly inhibited the progression of proteinuria in streptozotocin (STZ) induced diabetic rats at 0.3 and 1mg/kg, suggesting that this compound has potential to be a therapeutic agent for the treatment of diabetic nephropathy.

Synthesis and structure activity relationships of glycine amide derivatives as novel Vascular Adhesion Protein-1 inhibitors.

Vascular Adhesion Protein-1 (VAP-1) is a promising therapeutic target for the treatment of several inflammatory-related diseases including diabetic microvascular complication. We identified glycine amide derivative 3 as a novel structure with moderate VAP-1 inhibitory activity. Structure-activity relationship studies of glycine amide derivatives revealed that the tertiary amide moiety is important for stability in rat blood and that the position of substituents on the left phenyl ring plays an important role in VAP-1 inhibitory activity. We also found that low TPSA values and weak basicity are both important for high PAMPA values for glycine amide derivatives. These findings led to the identification of a series of orally active compounds with enhanced VAP-1 inhibitory activity. Of these compounds, 4g exhibited the most potent ex vivo efficacy, with plasma VAP-1 inhibitory activity of 60% after oral administration at 1mg/kg.

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.

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 (CL(int, 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 (CLu(int), (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 (F(a)F(g)) values were calculated from in vivo pharmacokinetic parameters in the literature (F(a)F(g) = 0.01-1.0). It was found that CLu(int, UGT) and human F(a)F(g) 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 F(a)F(g) regardless of UGT isoform.

Quantitative prediction of intestinal metabolism in humans from a simplified intestinal availability model and empirical scaling factor.

This study aimed to establish a practical and convenient method of predicting intestinal availability (F(g)) in humans for highly permeable compounds at the drug discovery stage, with a focus on CYP3A4-mediated metabolism. We constructed a "simplified F(g) model," described using only metabolic parameters, assuming that passive diffusion is dominant when permeability is high and that the effect of transporters in epithelial cells is negligible. Five substrates for CYP3A4 (alprazolam, amlodipine, clonazepam, midazolam, and nifedipine) and four for both CYP3A4 and P-glycoprotein (P-gp) (nicardipine, quinidine, tacrolimus, and verapamil) were used as model compounds. Observed fraction of drug absorbed (F(a)F(g)) values for these compounds were calculated from in vivo pharmacokinetic (PK) parameters, whereas in vitro intestinal intrinsic clearance (CL(int,intestine)) was determined using human intestinal microsomes. The CL(int,intestine) for the model compounds corrected with that of midazolam was defined as CL(m,index) and incorporated into a simplified F(g) model with empirical scaling factor. Regardless of whether the compound was a P-gp substrate, the F(a)F(g) could be reasonably fitted by the simplified F(g) model, and the value of the empirical scaling factor was well estimated. These results suggest that the effects of P-gp on F(a) and F(g) are substantially minor, at least in the case of highly permeable compounds. Furthermore, liver intrinsic clearance (CL(int,liver)) can be used as a surrogate index of intestinal metabolism based on the relationship between CL(int,liver) and CL(m,index). F(g) can be easily predicted using a simplified F(g) model with the empirical scaling factor, enabling more confident selection of drug candidates with desirable PK profiles in humans.

A comparison of pharmacokinetics between humans and monkeys.

To verify the availability of pharmacokinetic parameters in cynomolgus monkeys, hepatic availability (Fh) and the fraction absorbed multiplied by intestinal availability (FaFg) were evaluated to determine their contributions to absolute bioavailability (F) after intravenous and oral administrations. These results were compared with those for humans using 13 commercial drugs for which human pharmacokinetic parameters have been reported. In addition, in vitro studies of these drugs, including membrane permeability, intrinsic clearance, and p-glycoprotein affinity, were performed to classify the drugs on the basis of their pharmacokinetic properties. In the present study, monkeys had a markedly lower F than humans for 8 of 13 drugs. Although there were no obvious differences in Fh between humans and monkeys, a remarkable species difference in FaFg was observed. Subsequently, we compared the FaFg values for monkeys with the in vitro pharmacokinetic properties of each drug. No obvious FaFg differences were observed between humans and monkeys for drugs that undergo almost no in vivo metabolism. In contrast, low FaFg were observed in monkeys for drugs that undergo relatively high metabolism in monkeys. These results suggest that first-pass intestinal metabolism is greater in cynomolgus monkeys than in humans, and that bioavailability in cynomolgus monkeys after oral administration is unsuitable for predicting pharmacokinetics in humans. In addition, a rough correlation was also observed between in vitro metabolic stability and Fg in humans, possibly indicating the potential for Fg prediction in humans using only in vitro parameters after slight modification of the evaluation system for in vitro intestinal metabolism.

Prediction of oral absorption of griseofulvin, a BCS class II drug, based on GITA model: utilization of a more suitable medium for in-vitro dissolution study.

The in-vivo absorbability of drugs categorized into the biopharmaceutics classification system (BCS) class II is very difficult to be predicted because of the large variability in the absorption and/or dissolution kinetics and the lack of an adequate in-vitro system for evaluating the dissolution behavior. We tried to predict the in-vivo absorption kinetics of griseofulvin, categorized into BCS class II, orally administrated as powders into rats, based on Gastrointestinal-Transit-Absorption model (GITA model), consisting of the absorption, dissolution and GI-transit processes. Using the dissolution rate constants (k(dis)) of griseofulvin obtained with JP 1st solution, JP 2nd solution, FaSSIF, FeSSIF and modified SIBLM as a medium, simulation lines were not able to describe the observed mean plasma profile at all. On the other hand, a calculated line provided by employing k(dis) obtained with MREVID 2 (medium reflecting in-vivo dissolution 2), a new medium, was in better agreement with the observed mean plasma profile than existing media, indicating that the utilization of adequate k(dis) value made it possible to predict the in-vivo absorption kinetics of drugs classified into BCS class II based on GITA model and that MREVID 2 could be a useful medium for describing the in-vivo dissolution kinetics.

Analysis and prediction of absorption behavior of colon-targeted prodrug in rats by GI-transit-absorption model.

The gastrointestinal-transit-absorption (GITA) model is useful for the analysis and the prediction of the absorption behavior of drugs orally administered as solutions. In the present study, we tried to predict the plasma concentration-time profile of a colon-targeted prodrug, salicylazosulfanilic acid (SASA), and its parent drug, 5-aminosalicylic acid (5-ASA) which is regenerated after dosing. Prediction of plasma concentration-time profiles for SASA and 5-ASA was performed based on the GITA model using parameters describing GI-transit kinetics, the absorption in each GI segment, and the regeneration of 5-ASA in cecum. Plasma concentration-time profiles of both SASA and 5-ASA after oral administration of SASA were predicted very well by introducing a factor for the first-pass elimination of 5-ASA into the GITA model. The simulation study using the parameters obtained in the present study showed that about 94.7% of SASA reaches the cecum, where 5-ASA is regenerated very rapidly and 76.0% of 5-ASA is absorbed. Furthermore, the bioavailability of 5-ASA was estimated to be 0.330 because of the first-pass elimination through both cecum and liver. In conclusion, the absorption behaviors of a prodrug and its regenerated parent drug can be predicted very well and be clarified successfully using the GITA model.

Analysis and prediction of absorption behavior for theophylline orally administered as powders based on gastrointestinal-transit-absorption (Gita) model.

Absorption behavior of theophylline, categorized into Class I of Biopharmaceutics Classification System, orally administered as powders in rats was analyzed and predicted by Gastrointestinal-Transit-Absorption (GITA) model, which was modified to describe GI-transit kinetics and dissolution of powders orally administered. First of all, GI-transit kinetics of glass beads was examined to describe the transit kinetics of powders through GI tract in rats. The results showed that the gastric emptying of glass beads was slower than that of solution, but that there was not much difference in the transit rate constants through the small intestine and cecum between glass beads and solution. Furthermore, to introduce the dissolution process of theophylline powders into GITA model, an in-vitro dissolution test was examined for theophylline powders according to the Japanese Pharmacopoeia paddle method. The dissolution rate constants calculated based on the mean dissolution time were not so different in the range of pH from 1.2 to 6.5. Using the parameters for GI transit, dissolution and absorption obtained, the plasma concentration-time profile of theophylline after oral administration as powders to rats was predicted based on GITA model. The profile calculated was significantly correlated with the observed time course of plasma concentration for theophylline, and the parameters such as C(max) and AUC based on the predicted curve coincided with those on the observed data, showing that GITA model is useful for the prediction of the absorption behavior of drugs administered as powders. The simulation studies showed that about 80% of orally administered theophylline powders dissolved in the stomach and that the remaining powders rapidly moved to the lower jejunum and ileum, where they dissolved. Furthermore, it was suggested that theophylline is absorbed mostly in the upper small intestine, duodenum, upper jejunum and lower jejunum, after its oral administration as powders.