Prediction methods of drug-drug interactions of non-oral CYP3A4 substrates based on clinical interaction data after oral administrations - Validation with midazolam, alfentanil, and verapamil after intravenous administration and prediction for blonanserin transdermal patch.

Drug-drug interactions (DDI) have been examined for various drugs for oral use, but less for non-oral applications. This study provides DDI prediction methods for non-orally administered CYP3A4 substrates based on clinical DDI data of oral dosages. Gut availability (Fg) and fraction contribution of CYP3A4 to hepatic intrinsic clearance (fmCYP3A4) were predicted by AUC ratio (AUCR) in oral DDI study with/without grapefruit juice, and alteration in intrinsic clearances with/without ketoconazole, respectively. AUCRs of non-orally administered CYP3A4 substrates with/without inhibitors or inducers were predicted with the estimated Fg, fmCYP3A4 and changes in liver CYP3A4 activities with inhibitors/inducers predicted using Simcyp library. DDIs of intravenously administered midazolam and alfentanil with CYP3A4 inhibitors/inducers could be predicted well by this method with predicted AUCRs within ±64% of observed values. Moreover, maximum DDIs with strong CYP3A4 inducers could be predicted by comparing hepatic clearance with hepatic blood flow, as hepatic blood flow indicates the possible maximum hepatic clearance after strong enzyme induction. Predicted AUCRs of midazolam, alfentanil and R- and S-verapamil were less than, but not far from observed ratios, suggesting good conservative prediction. These methods were applied to blonanserin transdermal patch, suggesting much smaller interaction with CYP3A4 inhibitors/inducers compared to oral dosage of blonanserin.

Quantitative Prediction of Human Hepatic Clearance for P450 and Non-P450 Substrates from In Vivo Monkey Pharmacokinetics Study and In Vitro Metabolic Stability Tests Using Hepatocytes.

Accurate prediction of human pharmacokinetics for drugs remains challenging, especially for non-cytochrome P450 (P450) substrates. Hepatocytes might be suitable for predicting hepatic intrinsic clearance (CLint) of new chemical entities, because they can be applied to various compounds regardless of the metabolic enzymes. However, it was reported that hepatic CLint is underestimated in hepatocytes. The purpose of the present study was to confirm the predictability of human hepatic clearance for P450 and non-P450 substrates in hepatocytes and the utility of animal scaling factors for the prediction using hepatocytes. CLint values for 30 substrates of P450, UDP-glucuronosyltransferase, flavin-containing monooxygenase, esterases, reductases, and aldehyde oxidase in human microsomes, human S9 and human, rat, and monkey hepatocytes were estimated. Hepatocytes were incubated in serum of each species. Furthermore, CLint values in human hepatocytes were corrected with empirical, monkey, and rat scaling factors. CLint values in hepatocytes for most compounds were underestimated compared to observed values regardless of the metabolic enzyme, and the predictability was improved by using the scaling factors. The prediction using human hepatocytes corrected with monkey scaling factor showed the highest predictability for both P450 and non-P450 substrates among the predictions using liver microsomes, liver S9, and hepatocytes with or without scaling factors. CLint values by this method for 80% and 90% of all compounds were within 2- and 3-fold of observed values, respectively. This method is accurate and useful for estimating new chemical entities, with no need to care about cofactors, localization of metabolic enzymes, or protein binding in plasma and incubation mixture.

Time-dependent inhibition (TDI) of CYP1A2 by a CYP3A4-mediated reactive metabolite: proposal for a novel mechanism of irreversible TDI by a non-suicide substrate.

1. The purpose of this study was to clarify the mechanism of DSP-1053 time-dependent inhibition (TDI) for CYP1A2. 2. DSP-1053 inhibited time- and concentration-dependently CYP1A2 activity in human liver microsomes even in a dilution assay. However, DSP-1053 was not metabolized by recombinant human CYP1A2. These findings indicate that the inhibitory effect of DSP-1053 on CYP1A2 does not follow a general mechanism-based inhibition (MBI) because it did not seem to be a suicide substrate. 3. In fact, CYP1A2 was not inhibited with DSP-1053 pre-incubation in recombinant human CYP1A2. On the other hand, CYP1A2 was potently inhibited after pre-incubation with DSP-1053 in a mixture of human recombinant CYP1A2 and CYP3A4. In addition, DSP-1053 TDI of CYP1A2 in human liver microsomes was drastically reduced not only by addition of a CYP3A4 inhibitor, but also by addition of potassium cyanide (KCN), which is a trapping agent for iminium ions. We also confirmed in this study that CYP1A2 suicide inhibition by DSP-1053 metabolites generated by CYP3A4 had only minimal role in DSP-1053 TDI of CYP1A2. 4. In conclusion, a possible mechanism for DSP-1053 TDI of CYP1A2 is that DSP-1053 iminium ion, which is generated by CYP3A4, departs from CYP3A4 without inhibiting it and covalently binds to CYP1A2.

DSP-1053, a novel serotonin reuptake inhibitor with 5-HT1A partial agonistic activity, displays fast antidepressant effect with minimal undesirable effects in juvenile rats.

Enhancement of serotonergic neurotransmission has been the main stream of treatment for patients with depression. However, delayed therapeutic onset and undesirable side effects are major drawbacks for conventional serotonin reuptake inhibitors. Here, we show that DSP-1053, a novel serotonin reuptake inhibitor with 5-HT1A partial agonistic activity, displays fast antidepressant efficacy with minimal undesirable effects, especially nausea and emesis in animal models. DSP-1053 bound human serotonin transporter and 5-HT1A receptor with the K i values of 1.02 ± 0.06 and 5.05 ± 1.07 nmol/L, respectively. This compound inhibited the serotonin transporter with an IC50 value of 2.74 ± 0.41 nmol/L and had an intrinsic activity for 5-HT1A receptors of 70.0 ± 6.3%. In rat microdialysis, DSP-1053, given once at 3 and 10 mg kg(-1), dose-dependently increased extracellular 5-HT levels. In the rat forced swimming test, 2-week administration of DSR-1053 (1 mg kg(-1)) significantly reduced rats immobility time after treatment, whereas paroxetine (3 and 10 mg kg(-1)) required 3-week administration to reduce rats immobility time. In olfactory bulbectomy model, 1- and 2-week administration of DSP-1053 reduced both of emotional scores and activity in the open field, whereas paroxetine required 2 weeks to show similar beneficial effects. Although single administration of DSP-1053-induced emesis and vomiting in the rat and Suncus murinus, multiple treatment with this compound, but not with paroxetine, decreased the number of vomiting episodes. These results highlight the important role of 5-HT1A receptors in both the efficacy and tolerability of DSP-1053 as a new therapeutic option for the treatment of depression.

Hepatic, intestinal, renal, and plasma hydrolysis of prodrugs in human, cynomolgus monkey, dog, and rat: implications for in vitro-in vivo extrapolation of clearance of prodrugs.

Hydrolysis plays an important role in metabolic activation of prodrugs. In the current study, species and in vitro system differences in hepatic and extrahepatic hydrolysis were investigated for 11 prodrugs. Ten prodrugs in the data set are predominantly hydrolyzed by carboxylesterases (CES), whereas olmesartan medoxomil is also metabolized by carboxymethylenebutenolidase (CMBL) and paraoxonase. Metabolic stabilities were assessed in cryopreserved hepatocytes, liver S9 (LS9), intestinal S9 (IS9), kidney S9 (KS9), and plasma from human, monkey, dog, and rat. Of all the preclinical species investigated, monkey intrinsic hydrolysis clearance obtained in hepatocytes (CLint,hepatocytes) were the most comparable to human hepatocyte data. Perindopril and candesartan cilexetil showed the lowest and highest CLint,hepatocytes, respectively, regardless of the species investigated. Scaled intrinsic hydrolysis clearance obtained in LS9 were generally higher than CLint,hepatocytes in all species investigated, with the exception of dog. In the case of human and dog intestinal S9, hydrolysis intrinsic clearance could not be obtained for CES1 substrates, but hydrolysis for CES2 and CMBL substrates was detected in IS9 and KS9 from all species. Pronounced species differences were observed in plasma; hydrolysis of CES substrates was only evident in rat. Predictability of human hepatic intrinsic clearance (CLint,h) was assessed for eight CES1 substrates using hepatocytes and LS9; extrahepatic hydrolysis was not considered due to high stability of these prodrugs in intestinal and kidney S9. On average, predicted oral CLint,h from hepatocyte data represented 20% of the observed value; the underprediction was pronounced for high-clearance prodrugs, consistent with the predictability of cytochrome P450/conjugation clearance from this system. Prediction bias was less apparent with LS9, in particular for high-clearance prodrugs, highlighting the application of this in vitro system for investigation of prodrugs.

Species differences in hepatic and intestinal metabolic activities for 43 human cytochrome P450 substrates between humans and rats or dogs.

1. Prediction of human pharmacokinetics might be made more precise by using species with similar metabolic activities to humans. We had previously reported the species differences in intestinal and hepatic metabolic activities of 43 cytochrome P450 (CYP) substrates between cynomolgus monkeys and humans. However, the species differences between humans and rats or dogs had not yet been determined using comparable data sets with sufficient number of compounds. 2. Here, we investigated metabolic stabilities in intestinal and liver microsomes obtained from rats, dogs and humans using 43 substrates of human CYP1A2, CYP2J2, CYP2C, CYP2D6 and CYP3A. 3. Hepatic intrinsic clearance (CLint) values for most compounds in dogs were comparable to those in humans (within 10-fold), whereas in rats, those for the human CYP2D6 substrates were much higher and showed low correlation with humans. In dog intestine, as with human intestine, CLint values for almost all human CYP1A2, CYP2C, CYP2D6 substrates were not determined because they were very low. Intestinal CLint values for human CYP3A substrates in rats and dogs appeared to be lower for most of the compounds and showed moderate correlation with those in humans. 4. In conclusion, dogs showed the most similar metabolic activity to humans.

Significance of reductive metabolism in human intestine and quantitative prediction of intestinal first-pass metabolism by cytosolic reductive enzymes.

The number of new drug candidates that are cleared via non-cytochrome P450 (P450) enzymes has increased. However, unlike oxidation by P450, the roles of reductive enzymes are less understood. The metabolism in intestine is especially not well known. The purposes of this study were to investigate the significance of reductive metabolism in human intestine, and to establish a quantitative prediction method of intestinal first-pass metabolism by cytosolic reductive enzymes, using haloperidol, mebendazole, and ziprasidone. First, we estimated the metabolic activities for these compounds in intestine and liver using subcellular fractions. Metabolic activities were detected in human intestinal cytosol (HIC) for all three compounds, and the intrinsic clearance values were higher than those in human liver cytosol for haloperidol and mebendazole. These metabolic activities in HIC were NADPH- and/or NADH-dependent. Furthermore, the metabolic activities for all three compounds in HIC were largely inhibited by menadione, which has been used as a carbonyl reductase (CBR)-selective chemical inhibitor. Therefore, considering subcellular location, cofactor requirement, and chemical inhibition, these compounds might be metabolized by CBRs in human intestine. Subsequently, we tried to quantitatively predict intestinal availability (F(g)) for these compounds using human intestinal S9 (HIS9). Our prediction model using apparent permeability of parallel artificial membrane permeability assay and metabolic activities in HIS9 could predict F(g) in humans for the three compounds well. In conclusion, CBRs might have higher metabolic activities in human intestine than in human liver. Furthermore, our prediction method of human F(g) using HIS9 is applicable to substrates of cytosolic reductive enzymes.

Prediction of the intestinal first-pass metabolism of CYP3A and UGT substrates in humans from in vitro data.

This study aimed to establish a practical and simplified method of predicting intestinal availability in humans (F(g,human)) at the drug discovery stage using in vitro metabolic clearance values and permeability clearance values. A prediction model for F(g,human) of 19 CYP3A substrates and 5 UGT substrates was constructed based on the concept that the permeability clearance values mean the permeability across the basal membrane with a pH of 7.4 on both sides. Permeability clearance values were obtained by parallel artificial membrane permeability assay (PAMPA) at pH 7.4. PAMPA is widely used in the pharmaceutical industry as the earliest primary screening stage and enables estimation of the kinetics of transport by passive diffusion. For CYP3A substrates, the metabolic clearance was obtained from in vitro intrinsic clearance values in human intestinal or hepatic microsomes (CL(int,HIM) or CL(int,HLM), respectively). Using metabolic clearances corrected by the ratio of CL(int,HIM) to CL(int,HLM), HLM showed equivalent predictability to that of HIM for CYP3A substrates. For UGT substrates, the clearance was obtained from alamethicin-activated HIM using one incubation with both NADPH and UDPGA cofactors. The method proposed in this study could predict F(g,human) for the compounds investigated and represents a simplified method based on a new concept applicable to lower permeability compounds.

Species differences in intestinal metabolic activities of cytochrome P450 isoforms between cynomolgus monkeys and humans.

The oral bioavailability of some drugs is markedly lower in cynomolgus monkeys than in humans. One of the reasons for the low bioavailability in cynomolgus monkeys may be the higher metabolic activity of intestinal CYP3A; however, the species differences in intestinal metabolic activities of other CYP isoforms between cynomolgus monkeys and humans are not well known. In the present study, we investigated the intrinsic clearance (CL(int)) values in pooled intestinal microsomes from cynomolgus monkeys and humans using 25 substrates of human CYP1A2, CYP2J2, CYP2C, and CYP2D6. As in humans, intestinal CL(int) values of human CYP1A2 and CYP2D6 substrates in cynomolgus monkeys were low. On the other hand, intestinal CL(int) values of human CYP2J2 and CYP2C substrates in cynomolgus monkeys were greatly higher than those in humans. Using immunoinhibitory antibodies and chemical inhibitors, we showed that the higher intestinal CL(int) values of the human CYP2J2 and CYP2C substrates in cynomolgus monkeys might be caused by monkey CYP4F and CYP2C subfamily members, respectively. In conclusion, there is a possibility that the greatly higher metabolic activity of CYP2C and CYP4F in cynomolgus monkey intestine is one of the causes of the species difference of intestinal first-pass metabolism between cynomolgus monkeys and humans.

Prediction of the intestinal first-pass metabolism of CYP3A substrates in humans using cynomolgus monkeys.

To select high bioavailability compounds, it is necessary to predict the first-pass metabolism in the intestine. However, in vitro-in vivo predictions of the intestinal metabolism have proven both challenging and less definitive. The purpose of this study was to investigate prediction of intestinal first-pass metabolism in humans using cynomolgus monkeys. First, we investigated intrinsic metabolic activities in intestinal microsomes of monkeys (MIM) and humans (HIM) (CL(int, MIM) and CL(int, HIM), respectively) of 18 CYP3A substrates. The CL(int, MIM) values were found to be relatively high and showed excellent correlation with the CL(int, HIM) values. Subsequently, we determined the plasma concentrations of 9 CYP3A substrates (buspirone, carbamazepine, diazepam, felodipine, midazolam, nicardipine, nifedipine, saquinavir, and verapamil) in monkeys after an oral dose of 2 mg/kg with or without an oral dose of 5 mg/kg ketoconazole and calculated AUC((+vehicle))/AUC((+ketoconazole)), defined as F(g, monkey(observed)); we confirmed that the dose of ketoconazole inhibited only intestinal CYP3A metabolism by preliminary in vitro and in vivo experiments using ketoconazole. The F(g, monkey(observed)) was lower than the F(g, human(observed)) for most compounds, but moderate correlation was observed. Furthermore, using these data, we established a new methodology to estimate F(g, human(predicted)) more precisely on the basis of the assumption that intestinal physiological conditions other than intrinsic metabolic activity would be the same between monkeys and humans. In conclusion, the in vivo model using cynomolgus monkeys in this study is useful for prediction of intestinal first-pass metabolism by CYP3A in humans because it was able to predict F(g, human) of all nine compounds investigated.

Inhibitory effects of various beverages on human recombinant sulfotransferase isoforms SULT1A1 and SULT1A3.

Sulfotransferase (SULT) 1A1 and SULT1A3 play important roles in the presystemic inactivation of beta(2) agonists in the liver and intestine, respectively. The study aimed to investigate the inhibitory effects of grapefruit juice, orange juice, green tea, black tea and oolong tea and their constituents on the activities of SULT1A1 and SULT1A3. The activities of both SULT1A1 and SULT1A3 were significantly inhibited by all the beverages investigated at a concentration of 10%. The beverage constituents were tested in concentration ranges considered to be physiologically relevant. The grapefruit constituent, quercetin, completely inhibited SULT1A1, while quercetin and naringin both partially inhibited SULT1A3. The orange constituents, tangeretin and nobiletin, also completely inhibited SULT1A1. The tea constituents, (-)-epicatechin gallate and (-)-epigallocatechin gallate, both almost completely inhibited SULT1A1 and SULT1A3. Moreover, the theaflavin and thearubigin fractions of black tea both completely inhibited SULT1A1 and strongly inhibited SULT1A3. The inhibitory action of green tea on SULT1A3 was competitive, while that of black tea and oolong tea was mixed competitive/non-competitive. Mechanism-based inhibition was not observed with any beverage. In conclusion, various beverages, especially teas, inhibit the function of SULT1A3, and therefore may have the potential to increase the bioavailability of orally administered substrates of SULT1A3, such as beta(2) agonists.

Inhibitory effects of various beverages on ritodrine sulfation by recombinant human sulfotransferase isoforms SULT1A1 and SULT1A3.

PURPOSE: Ritodrine is known to undergo extensive presystemic sulfation in the intestinal mucosa, and its bioavailability is as low as 30%. Accordingly, inhibition of intestinal sulfation may lead to an increase in the bioavailability of ritodrine. In this study, we aimed to investigate the activities of ritodrine sulfation by SULT1A1, which is expressed predominantly in the liver, and SULT1A3, which is expressed predominantly in the intestine, as well as the inhibitory effects of beverages on their activities. METHODS: We investigated ritodrine sulfation by using recombinant human sulfotransferase (SULT) 1A1 and SULT1A3 in an in vitro study. Next, we investigated the inhibitory effects of grapefruit juice, orange juice, green tea, and black tea on ritodrine sulfation. RESULTS: Sulfation of ritodrine by SULT1A3 was much higher than that by SULT1A1, suggesting that the bioavailability of ritodrine may be limited by intestinal SULT1A3. The ritodrine sulfation activities of SULT1A1 and SULT1A3 were significantly inhibited by all beverages examined at a concentration of 10%. Green tea and black tea exhibited potent inhibition; even at a concentration of 5%, they both inhibited SULT1A1 by 100% and SULT1A3 by >or=95%. CONCLUSION: Our results suggest that concomitant ingestion of beverages such as green tea and black tea may increase the bioavailability of orally administered ritodrine, and perhaps other beta2-agonists, and lead to an increase in the clinical effects or adverse reactions.