Evaluation of QSAR models for predicting mutagenicity: outcome of the Second Ames/QSAR international challenge project.

Quantitative structure-activity relationship (QSAR) models are powerful in silico tools for predicting the mutagenicity of unstable compounds, impurities and metabolites that are difficult to examine using the Ames test. Ideally, Ames/QSAR models for regulatory use should demonstrate high sensitivity, low false-negative rate and wide coverage of chemical space. To promote superior model development, the Division of Genetics and Mutagenesis, National Institute of Health Sciences, Japan (DGM/NIHS), conducted the Second Ames/QSAR International Challenge Project (2020-2022) as a successor to the First Project (2014-2017), with 21 teams from 11 countries participating. The DGM/NIHS provided a curated training dataset of approximately 12,000 chemicals and a trial dataset of approximately 1,600 chemicals, and each participating team predicted the Ames mutagenicity of each trial chemical using various Ames/QSAR models. The DGM/NIHS then provided the Ames test results for trial chemicals to assist in model improvement. Although overall model performance on the Second Project was not superior to that on the First, models from the eight teams participating in both projects achieved higher sensitivity than models from teams participating in only the Second Project. Thus, these evaluations have facilitated the development of QSAR models.

Predictive performance of three practical approaches for grapefruit juice-induced 2-fold or greater increases in AUC of concomitantly administered drugs.

WHAT IS KNOWN AND OBJECTIVE: Clinical pharmacists have a challenging task when answering patients' question about whether they can take specific drugs with grapefruit juice (GFJ) without risk of drug interaction. To identify the most practicable method for predicting clinically relevant changes in plasma concentrations of orally administered drugs caused by the ingestion of GFJ, we compared the predictive performance of three methods using data obtained from the literature. METHODS: We undertook a systematic search of drug interactions associated with GFJ using MEDLINE and the Metabolism & Transport Drug Interaction Database (DIDB version 4.0). We considered an elevation of the area under the plasma concentration-time curve (AUC) of 2 or greater relative to the control value [AUC ratio (AUCR) ≥ 2.0] as a clinically significant interaction. RESULTS AND DISCUSSION: The data from 74 drugs (194 data sets) were analysed. When the reported information of CYP3A involvement in the metabolism of a drug of interest was adopted as a predictive criterion for GFJ-drug interaction, the performance assessed by positive predictive value (PPV) was low (0.26), but that assessed by negative predictive value (NPV) and sensitivity was high (1.00 for both). When the reported oral bioavailability of ≤ 0.1 was used as a criterion, the PPV improved to 0.50 with an acceptable NPV of 0.81, but sensitivity was reduced to 0.21. When the reported AUCR was ≥ 10 after co-administration of a typical CYP3A inhibitor, the corresponding values were 0.64, 0.79 and 0.19, respectively. WHAT IS NEW AND CONCLUSION: We consider that an oral bioavailability of ≤ 0.1 or an AUCR of ≥ 10 caused by a CYP3A inhibitor of a drug of interest may be a practical prediction criterion for avoiding significant interactions with GFJ. Information about the involvement of CYP3A in their metabolism should also be taken into account for drugs with narrow therapeutic ranges.

Reliability of predicted renal function in Japanese patients on cisplatin therapy.

Cisplatin, cis-Dichlorodiammine platinum (II) (CDDP) remains a major antineoplastic drug for the treatment of solid tumors. Its chief dose-limiting side effect is nephrotoxicity. To make a safe and effective dosing regimen of a drug excreted mainly by the renal route, evaluation of patients' renal function is essential. Creatinine clearance (CLcr) or glomerular filtration rate (GFR) is considered to be a standard renal-function test. Several equations have been used in clinical settings, to predict CLcr and GFR using serum creatinine concentration. We carried out a retrospective analysis of the correlation between 24-hour CLcr measured by a urine collection method; and the predicted CLcr and GFR estimated by various equations such as Jelliffe, Yasuda, Orita, Mawer, Mawer, MDRD and modified MDRD, and Cockcroft-Gault. This study used data from Japanese head-and-neck cancer patients, before and after chemotherapy with CDDP. Slopes of regression lines of scatter plots between measured CLcr and predicted renal function in post-CDDP patients were less compared to pre-CDDP patients. On the other hand, Y-intercepts were noted in the scatter plots on renal function from all equations. These results suggest that evaluation of renal function using predictive formulae may have been over-/under-estimated after CDDP administration.

Quantitative structure-interaction relationship analysis of 1,4-dihydropyridine drugs in concomitant administration with grapefruit juice.

Quantitative structure-interaction relationship (QSIR) analyses of 1,4-dihydropyridine drugs were performed on grapefruit juice interaction potentials to characterize the interaction and evaluate drugs not yet tested in clinical research. AUC ratios of drugs with and without grapefruit juice ingestion were estimated as grapefruit juice interaction potentials from clinical studies on dihydropyridine drugs such as amlodipine, azelnidipine, benidipine, cilnidipine, felodipine, manidipine, nicardipine, nifedipine, nimodipine, nisoldipine, and pranidipine. The minimal energy conformation in each dihydropyridine drug was searched for using Merck Molecular Force Field (MMFFaq), and then geometry optimization was performed by density-functional-theory (DFT) calculation (B3LYP/6-31G**). The geometric, electronic, and physicochemical features including molecular size, dipole moment, total energy, HOMO/LUMO energies, and logP values were then obtained. Dragon descriptors were also calculated by optimized 3D-structures. The relation between the potentials and over 1000 of the molecular properties was investigated using statistical techniques including partial least squares analysis with genetic algorithm (GA-PLS) to a variable subset selection. Some PLS regression equations including logP values and dragon descriptors as explanatory variables were constructed in which the maximal contribution coefficient was 94%. These models could be applied to estimate the interaction potentials of other dihydropyridine drugs that have gone unreported in interactions with drugs such as aranidipine, barnidipine, clevidipine, lemildipine, lercanidipine, niguldipine, niludipine, and nilvadipine. In the assessment of major dihydropyridines, amlodipine was found to be the safest drug to avoid interactions among the drugs investigated in the present study.

Construction of a model to estimate the CYP3A inhibitory effect of grapefruit juice.

Grapefruit juice (GFJ) is known to affect the pharmacokinetics of a variety of drugs administered concomitantly and this is due to inhibition of intestinal CYP3A, a barrier protein for drug absorption. Some compounds such as furanocoumarin derivatives have been reported as inhibitors of the enzyme. On the other hand, inhibitory potentials of GFJ on CYP3A-oxidation activities differ widely between brands of juices. Information on the percentage contributed by ingredients in GFJ is also limited. Therefore, construction of prediction models for the CYP3A inhibitory potentials of GFJ brands was attempted by using concentrations of ingredients in GFJ. Concentrations of bergaptol, bergamottin, 6', 7'-dihydroxybergamottin, naringin, and naringenin in 23 kinds of GFJ were determined with high-performance liquid chromatography (HPLC). Furthermore, inhibitory effects on CYP3A activity were measured based on the initial rate for testosterone 6beta-hydroxylation in the presence of each GFJ. Results of multi-regression analyses between the ingredients and the enzymatic inhibitory effects revealed that concentrations of bergamottin, 6',7'-dihydroxybergamottin, and naringin were significant variables for CYP3A inhibition of GFJ. According to the standard partial regression coefficient for each explanatory variable, bergamottin and 6',7'-dihydroxybergamottin are the most important factors for inhibition. The multiple correlation coefficient (R) and the multiple correlation coefficient with leave-one-out cross validation (Q) of the model equation were 0.94 and 0.91, respectively. These results suggest that the concentrations of ingredients can explain most variances of inhibitory effects among brands. This model may be a useful method for the prediction of the GFJ interaction potential.

Publication bias on clinical studies of pharmacokinetic interactions between felodipine and grapefruit juice.

Plasma concentrations of a variety of drugs are known to be increased by concomitant administration of grapefruit juice (GFJ) when the drugs are administered orally. Dihydropyridine Ca channel blockers, that form one of the major categories of antihypertensive, have been studied for interactions for the longest time in research history. Especially, felodipine has been the most studied dihydropyridine drug. Although a lot of clinical research has been performed on the pharmacokinetic variations of felodipine, there has been no adequate systematic study. Therefore, publications related to felodipine-GFJ interactions were integrated and analyzed with statistical procedures of meta-analysis to characterize these clinical studies. Furthermore, funnel plots were created to validate publication bias in the data. Integration of AUC values on GFJ-administered and control groups in 12 publications revealed that felodipine is apparently affected by interaction. However, publication bias was observed in the funnel plots, and null hypothesis of no bias was rejected by Begg's test. These findings suggest that the pharmacokinetic interactions with GFJ might be overrated in the fundamental trial stage.

Prediction models for feverishness developed during interferon therapy of chronic hepatitis C patients.

Pegylated interferon (peginterferon) and ribavirin combination therapy is used extensively for therapy of chronic hepatitis C. Most patients that receive this therapy are known to develop influenza-like symptoms with fever and headache. Therefore, we attempted to construct a multiple-regression model to predict the intensity of feverishness from the profiles of such patients. A retrospective survey of the medical charts of patients with chronic hepatitis C that have been on peginterferon-alpha-2b and ribavirin combination therapy was performed. Body temperatures of patients at 8.5 h after receiving interferon injection on day one of therapy were the objective variables. Patients' profiles such as sex, age, and blood test results before the injection were defined as explanatory variables. Genetic algorism with leave-one-out cross-validation as selection pressure was applied in the selection of variables. The final model for prediction was determined by bootstrap validation. As a result, a significant multiple-regression model including sex, BUN, and leukocyte count as descriptors was constructed. The prediction of patients with severe fever in the model equation is of some help regarding the proper use of antipyretics in interferon therapy.

Quantitative structure-activity relationship (QSAR) analysis of the inhibitory effects of furanocoumarin derivatives on cytochrome P450 3A activities.

Furanocoumarin derivatives (FCs) present in grapefruit and other plants cause pharmacokinetic interactions such as increased absorption of various drugs because the constituents have inhibitory effects on drug metabolizing activities of cytochrome P450 (CYP) 3A that is expressed in intestinal mucosal cells. Though it has been 20 years since such an interaction was discovered, little is still known about the relationship between the molecular characters of FCs and their inhibitory effects. Therefore, the chemical and physicochemical characterizations of the biological activities of FCs were examined by quantitative structure-activity relationship (QSAR) analysis using 37 types of FCs. Common logarithmic IC50 values of human liver microsomal testosterone 6beta-hydroxylations were configured as objective variables. A variety of structural, physicochemical, and quantum chemical descriptors were calculated from 2D and optimized 3D structures in the 37 FCs as explanatory variables. Simple and multiple linear regression analyses were used to evaluate these parameters. Constructed regression models were validated with leave-one-out cross validation and applicable regression diagnostic methods. As a result, logP value, molecular volume, molecular weight, molecular surface area, polar surface area, minimal electrostatic potential, formation energy, and homo energy of each FC were significantly related with the logIC50 value. These relationships indicate that molecular characteristics including lipophilicity, molecular size, electrostatic stabilization, and electron-donating ability of FCs can control FC-CYP interactions. These findings could be useful to predict CYP inhibitory effects of other FCs in foods, drinks, and other natural products such as grapefruit juice and herbal drugs.

White and colored grapefruit juice produce similar pharmacokinetic interactions.

Colored (pink and red) grapefruit pulp contains lower amounts of the furanocoumarin derivatives that cause pharmacokinetic interactions than white grapefruit pulp. However, few studies have examined interactions with colored juice products. Therefore, we examined the potential interactions of both white and colored grapefruit products by measuring the concentrations of furanocoumarin derivatives and inhibition of the metabolizing cytochrome P450 (CYP) 3A enzymes, the target of the furanocoumarins. We measured concentrations of three major furanocoumarin derivatives, bergaptol, bergamottin, and 6',7'-dihydroxybergamottin, with high-performance liquid chromatography in 21 brands of grapefruit juice sold in Japan, including 14 white and 7 colored brands. The mean difference in bergaptol, bergamottin, and 6',7'-dihydroxybergamottin concentrations in white grapefruit juice samples was 1.59, 0.902, and 1.03 times, respectively, the amounts in colored samples. White samples inhibited CYP3A-mediated testosterone-6beta oxidation in human liver microsomes by 1.04 and 0.922 times (whole juice and furanocoumarin, respectively) the inhibition by colored juice. Thus, colored grapefruit juice may produce drug interactions at the same rate as white grapefruit juice.

Determination of bergamottin in human plasma after grapefruit juice ingestion by an UPLC/MS/MS method.

Bergamottin was identified as a cause of pharmacokinetic interaction with grapefruit juice intake and as a physiologically active substance involved in lipolysis. However, the quantification method on concentrations of bergamottin in systemic circulation has not been well established. The aim of this study was to develop a simple, sensitive and high-throughput determination system for bergamottin in human plasma using an ultra performance liquid chromatography (UPLC)-MS-MS method. The UPLC system equipped with a UPLC BEH C18 column (2.1 x 50 mm, 1.7 microm) and an ESI prove was appropriate for detection of bergamottin. As a result, a primary product ion (m/z = 203) from precursor ion of bergamottin (m/z = 339) was observed. Plasma from a human volunteer who consumed grapefruit juice one hour before the time of blood sampling, was measured with the UPLC/MS/MS system. The determination of plasma-bergamottin was performed with the highest sensitivity presently available. In conclusion, we succeeded high performance bergamottin-determination in human plasma after grapefruit juice ingestion. The procedure can be usefulto clarify pharmacokinetic and pharmacodynamic characteristics of bergamottin.

Drug interaction potentials among different brands of grapefruit juice.

The discrepancy of drug-interaction potential among different brands of grapefruit juice was estimated based on inhibition of CYP3A activity caused by furanocoumarin derivatives in the grapefruit juice. Heat treatment of the grapefruit juice at 95 degrees C for 1 h was utilized to degrade the furanocoumarins. Initial velocity of testosterone 6beta-oxidation using human liver microsomes was determined as an indicator of the CYP3A activities. Changes in the velocities of the reaction mixture were observed when 10% of each brand of untreated grapefruit juice or heat-treated grapefruit juice was added. The differences in the velocities between untreated and heat-treated grapefruit juice were defined as the potentials of furanocoumarin-caused CYP3A-inhibitions.

Metabolism of bucolome in rats. Stability and biliary excretion of bucolome N-glucuronide.

Bucolome (BCP) is a non-steroidal anti-inflammatory drug, which is used in the treatment of chronic articular rheumatism. Bucolome N-glucuronide (BCP-NG), a metabolite of BCP, is the first unique N-glucuronide of barbituric acid derivatives. First, the stability of BCP-NG in various pH aqueous solutions was studied. BCP-NG was quite unstable under neutral and acidic conditions, and is easily hydrolyzed to BCP. Based on these characteristics of BCP-NG, a simple, rapid and highly sensitive method for the simultaneous determination of BCP and BCP-NG with phenylbutazone (I.S.) in biological fluids was developed using high-performance liquid chromatography (HPLC). A reversed-phase ODS column was used for the separation of BCP, BCP-NG and I.S. A pharmacokinetic study for BCP and BCP-NG was carried out in male Wistar/ST rats following i.v. administration of BCP at a dose of 10 mg/kg body weight. The slow plasma elimination of BCP with time was shown. A major metabolite of BCP in bile was N-glucuronide. The cumulative amounts of BCP and BCP-NG in the bile over 8 h were approximately 2.4 +/- 1.4% and 12.6 +/- 2.3% of the dose, respectively. BCP and BCP-NG in the urine were 2.7 +/- 0.7% and 3.2 +/- 0.3% of the dose. Although BCP had a long half-life (over 8.5 h), the preliminary pharmacokinetic parameters (0-8 h) were determined: t 1/2, 8.52 +/- 1.96 h; AUC, 419.9 +/- 45.2 microg x h/ml; MRT, 3.29 +/- 0.11 h; CLtot, 5.93 +/- 0.54 ml/h; and Vdss, 19.5 +/- 1.3 l. These observations are the first pharmacokinetic findings for the N-glucuronide of the barbituric acid derivatives.

Effects of furanocoumarin derivatives in grapefruit juice on nifedipine pharmacokinetics in rats.

PURPOSE: It has been reported that grapefruit juice (GJ) causes a pharmacokinetic interaction with many drugs after co-ingestion. It is postulated that the substances in GJ may inhibit the first-pass metabolism during the intestinal absorption process. In recent years, several furanocoumarin derivatives that inhibit P450 activity in intestinal microsomes were isolated from GJ. In this study, we report the effects of the furanocoumarin derivatives in GJ on the nifedipine (NFP) pharmacokinetics in rats. METHODS: Three furanocoumarin derivatives (bergaptol [BT], bergamottin [BG], and 6',7'-dihydroxybergamottin [DHB]) found in GJ were used in this study. Each furanocoumarin was reconstituted in orange juice at the same concentration as in the GJ. Two milliliters of each sample was administered into the rat duodenum. After 30 min, NFP was intraduodenally administered at a dose of 3 mg/kg body weight. The NFP concentrations in the plasma samples were determined by HPLC. RESULTS: A significant increase in the AUC of NFP was observed only in the rats administered BG; 1.5 times that of the control group. The result was quite identical with that of the group that was administered GJ. BT and DHB had no significant effects on the NFP pharmacokinetics. CONCLUSIONS: The results strongly suggested that BG in GJ might be the substance that elevates the NFP plasma concentrations.

Effects of long-term grapefruit juice ingestion on nifedipine pharmacokinetics: induction of rat hepatic P-450 by grapefruit juice.

We studied the effects of short- and long-term ingestion of grapefruit juice (GJ) on nifedipine (NFP) pharmacokinetics in rats. Thirty minutes after intraduodenal (id) administration of 2.0 ml of GJ or saline, NFP was i.v. or id administered at a dose of 3 mg/kg b. wt. No significant differences were observed in pharmacokinetic values between the two groups after i.v. administrations of NFP. By contrast, after id administration, the mean AUC value in the GJ group was approximately 1.62 times that in the control group, and the mean apparent clearance (CL) decreased by approximately 40%. In addition, 2.0 ml of GJ was orally administered twice a day (9:00 AM and 7:00 PM) for 10 consecutive days; on the 11th day the pharmacokinetics of NFP were examined again. Irrespective of route of administration (i.v. or id), NFP CL from plasma in these GJ-treated rats was considerably faster than that in the rats treated with GJ for a short (30-min) period. In microsomes prepared from the intestinal mucosa of animals receiving long-term administration of GJ, NFP oxidation activity (0.21 +/- 0.02 nmol/mg/min) and P-450 content (0.045 +/- 0.009 nmol/mg) were significantly lower than those in untreated rats (0.32 +/- 0.05 nmol/mg/min and 0.060 +/- 0.007 nmol/mg). In hepatic microsomes from the same rats, however, NFP oxidation activity (1.43 +/- 0.17 nmol/mg/min) and P-450 content (0.66 +/- 0.07 nmol/mg) were distinctly greater than those in untreated rats (1.00 +/- 0.06 nmol/mg/min and 0.51 +/- 0.04 nmol/mg). In conclusion, short-term id exposure to GJ resulted in increased NFP bioavailability, whereas long-term administration of GJ resulted in reduced bioavailability and increased CL.

Ion-pair reversed-phase high-performance liquid chromatography of adenine nucleotides and nucleoside using triethylamine as a counterion.

An isocratic HPLC method for the simple and selective determination of adenine nucleoside and nucleotides has been developed. The separation is achieved at room temperature by reversed-phase chromatography (Shiseido, Capcell Pak C18). A mixture of 0.1 M triethylamine (TEA) phosphate buffer and methanol (95:5, v/v) is used as a standard eluent. Influence of pH and concentrations of organic modifiers and TEA ion on capacity factors of adenine compounds has been investigated. It has been also found that the TEA ion in the eluent is adsorbed onto the reversed-phase surface. The results clearly demonstrate that ion-pair formation with TEA ion occurs probably both in the mobile phase and on the stationary phase and governs the retention of adenine and nucleotides in the present system. The HPLC system is applied to the analysis of adenine nucleotides formed as intermediates in the synthesis of 3'-phosphoadenosine 5'-phosphosulphate (PAPS) and to the assays of ATPases and 5'-nucleotidase activities in rat liver plasma membrane. This method is a new type of ion-pair reversed-phase HPLC system and is suitable for the separation of highly polar organic anions, especially for adenine nucleotides.

Heat-induced DNA cleavage by esperamicin antitumor antibiotics.

Esperamicin A1 effectively breaks DNA strands upon heating at 50 degrees C. The preferential DNA cutting sites of heat-activated esperamicin A1 are random and clearly differ from those of thiol- or UV-light-mediated DNA breakage with esperamicin A1. The absence of heat-induced DNA cleavage by esperamicin Z and the induction of the DNA breakage by esperamicin A1 disulfide indicate that (1) the enediyne core plays a significant role in this DNA strand scission and (2) the DNA cutting with the heat-activated esperamicin antibiotics does not necessarily require a trisulfide trigger in the aglycon portion. On the basis of the present results, a probable mechanism for the heat-induced DNA cleavage of esperamicin A1 has been proposed.

Light-induced DNA cleavage by esperamicin and neocarzinostatin.

Ultraviolet radiation of the enediyne drugs is effective in causing nicks in supercoiled DNA. Of special interest is the fact that the observed nucleotide cleaving specificity for the UV light- and thiol-activated antibiotics was the same with esperamicin A1, but was different with neocarzinostatin. In addition to the preferred cutting of T and A bases, the light-activated neocarzinostatin attacked certain G bases which were rarely cleaved by the thiol-activated neocarzinostatin. It should be noted that these enediyne antibiotics lose the DNA breakage activity after light-exposure for 30 min.

Nucleotide-specific cleavage and minor-groove interaction of DNA with esperamicin antitumor antibiotics.

The cleavage of DNA by esperamicin is greatly accelerated in the presence of thiol compounds. Oxygen and active oxygen-radical scavengers have no significant influence upon DNA strand breakage by esperamicin. The preferential cutting sites of esperamicin are at thymidylate residues, and the frequency of bases attacked (T greater than C greater than A greater than G) is different from that of calicheamicin (C much greater than T greater than A = G), neocarzinostatin (T greater than A greater than C greater than G), or bleomycin (C greater than T greater than A greater than G). Esperamicin preferentially attacks at T and C bases in oligopyrimidine sequences such as 5'-CTC-3', 5'-TTC-3', and 5'-TTT-3'. In contrast to the preferred sites of cleavage by bleomycin, 5'-GT-3' and 5'-GC-3', the preferred sites of esperamicin-mediated DNA degradation are 5'-TG-3' and 5'-CG-3' sequences. The nucleotide-specific cleavage mode of esperamicin is significantly affected by pretreatment of DNA with netropsin and distamycin A, suggesting that interaction of esperamicin occurs through the minor groove of B-DNA. This is further supported by the asymmetric cleavage pattern to the 3' side on the opposite strand of the DNA. The roles of the fucose-anthranilate moiety and the trisaccharide side chain of esperamicin in DNA binding and base recognition are discussed.

Nucleotide specificity of DNA cleavage by esperamycin/calichemicin antitumor antibiotics.

Esperamycin shows potent DNA cleavage activity in the presence of sulfhydryl-containing compounds such as glutathione and dithiothreitol. In the esperamycin-mediated DNA degradation, deoxythymidylic and deoxycytidylic acid residues at oligopyrimidine regions of 5'-CTC, TTC, and TTT sequences are preferred cleavage sites. The DNA binding of a typical A-T specific minor groove binder, netropsin or distamycin A, strongly inhibits the DNA cuttings at 5'-TTC, TTT, and AAA sequences.