Frank W. Lee

Millennium Pharmaceuticals Inc, Boston, Massachusetts, United States

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Publications (31)73.41 Total impact

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    ABSTRACT: Basic lipophilic compounds are characterized by a high volume of distribution as a result of extensive tissue uptake. The main mechanisms of the high tissue distribution have been proposed through the nonspecific binding to membrane phospholipids and the trapping by acidic subcellular compartments, mainly lysosomes. The present studies evaluated the effect of lysosomal trapping on drug tissue distribution and drug-drug interactions of weak bases in hepatocytes from various species and in Sprague-Dawley (SD) rats. The well-known lysosomal inhibitors (monensin and chloroquine) decreased the accumulation of imipramine in hepatocytes from mouse, rat, dog, monkey, and human. In contrast, the inhibition of thioridazine accumulation by lysosomal inhibitors was only observed in mouse hepatocytes, but not in other tested species. In SD rats, lysosomal trapping contributed to the high tissue distribution of imipramine and thioridazine, suggested by the correlation between the rank orders of the tissue distributions with the rank orders of lysosome amount in tissues. Pre-treatment of chloroquine decreased the exposure of imipramine in various tissues, except liver. The heart had the most significant effect. However, co-administration of chloroquine did not change AUC of thioridazine in rat tissues except heart and liver. Thus, cultured hepatocytes can be used as an in vitro model to estimate the contribution of lysosomal trapping on tissue accumulation. The rat tissue distribution study also indicates that ysosomal trapping mediated drug-drug interaction (DDI) in tissues is possible. The inhibition of chloroquine on the clearance of imipramine and thioridazine might also contribute to these DDIs. The combined effects of CYP-mediated metabolism and lysosome trapping need to be further investigated.
    No preview · Conference Paper · Nov 2011
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    ABSTRACT: Tandutinib is a tyrosine kinase inhibitor under investigation for the treatment of solid and hematological tumors. We evaluated efflux transporter substrate specificity of tandutinib in Caco-2 cells, and the role of efflux transporters in the disposition of tandutinib in rats and efflux transporter knock-out mice. These studies demonstrated that tandutinib is a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in Caco-2 cells. In rats, administration of GF120918, before treatment with tandutinib orally resulted in approximately a seven-fold increase in the mean plasma area under the concentration-versus-time curve (AUC) compared to the vehicle control group. In mice, after intravenous administration of tandutinib, the mean plasma AUC values in the Bcrp1(-/-) mice and Mdr1a/b(-/-) mice was 1.53- and 1.20-fold greater than that of the wild type (WT) mice, respectively. After oral administration, the drug exposure in Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)/Bcrp1(-/-) mice was higher than in the WT mice. The brain to plasma exposure ratio (B/P) of tandutinib in Mdr1a/b(-/-) mice increased by 2- to 3-fold over that in the WT mice. There was a 13-fold increase in B/P in Mdr1a/b(-/-)/Bcrp1(-/-) mice. This finding illustrates that P-gp and Bcrp play a role in oral absorption, systemic clearance, and brain penetration of tandutinib in the rodents. P-gp affected oral absorption and brain penetration of tandutinib to a greater extent than Bcrp, but Bcrp contribution to systemic clearance of tandutinib was greater than P-gp. Thus, co-administration of efflux pump inhibitors may be a useful strategy to enhance tandutinib absorption and brain penetration clinically.
    No preview · Article · Dec 2010
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    ABSTRACT: Tandutinib is a tyrosine kinase inhibitor under investigation for the treatment of solid and hematological tumors. We evaluated efflux transporter substrate specificity of tandutinib in Caco-2 cells, and the role of efflux transporters in the disposition of tandutinib in rats and efflux transporter knock-out mice. These studies demonstrated that tandutinib is a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in Caco-2 cells. In rats, administration of GF120918, before treatment with tandutinib orally resulted in approximately a seven-fold increase in the mean plasma area under the concentration-versus-time curve (AUC) compared to the vehicle control group. In mice, after intravenous administration of tandutinib, the mean plasma AUC values in the Bcrp1(-/-) mice and Mdr1a/b(-/-) mice was 1.53- and 1.20-fold greater than that of the wild type (WT) mice, respectively. After oral administration, the drug exposure in Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)/Bcrp1(-/-) mice was higher than in the WT mice. The brain to plasma exposure ratio (B/P) of tandutinib in Mdr1a/b(-/-) mice increased by 2- to 3-fold over that in the WT mice. There was a 13-fold increase in B/P in Mdr1a/b(-/-)/Bcrp1(-/-) mice. This finding illustrates that P-gp and Bcrp play a role in oral absorption, systemic clearance, and brain penetration of tandutinib in the rodents. P-gp affected oral absorption and brain penetration of tandutinib to a greater extent than Bcrp, but Bcrp contribution to systemic clearance of tandutinib was greater than Pgp. Thus, co-administration of efflux pump inhibitors may be a useful strategy to enhance tandutinib absorption and brain penetration clinically.
    No preview · Article · Nov 2010 · Drug Metabolism Letters
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    ABSTRACT: Organic anion-transporting polypeptides (OATPs), members of the SLCO/SLC21 family, mediate the transport of various endo- and xenobiotics. In human liver, OATP1B1, 1B3, and 2B1 are located at the basolateral membrane of hepatocytes and are involved in hepatic drug uptake and biliary elimination. Clinically significant drug-drug interactions (DDIs) mediated by hepatic OATPs have drawn great attention from clinical practitioners and researchers. However, there are considerable challenges to prospectively understanding the extent of OATP-mediated DDIs because of the lack of specific OATP inhibitors or substrates and the limitations of in vitro tools. In the present study, a novel RNA interference knockdown sandwich-cultured human hepatocyte model was developed and validated. Quantitative polymerase chain reaction, microarray and immunoblotting analyses, along with uptake assays, illustrated that the expression and transport activity of hepatic OATPs were reduced by small interfering (siRNA) efficiently and specifically in this model. Although OATP siRNA decreased only 20 to 30% of the total uptake of cerivastatin into human hepatocytes, it caused a 50% reduction in cerivastatin metabolism, which was observed by monitoring the formation of the two major metabolites of cerivastatin. The results suggest that coadministration of a drug that is a hepatic OATP inhibitor could significantly alter the pharmacokinetic profile of cerivastatin in clinical studies. Further studies with this novel model demonstrated that OATP and cytochrome P450 have a synergistic effect on cerivastatin-gemfibrozil interactions. The siRNA knockdown sandwich-cultured human hepatocytes may provide a new powerful model for evaluating DDIs.
    Preview · Article · Sep 2010 · Drug metabolism and disposition: the biological fate of chemicals
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    ABSTRACT: MLN4924 is a potent and selective inhibitor of Nedd8-Activating Enzyme (NAE), a novel target for anticancer therapy. NAE is considered to play a key role in protein homeostasis disruption of which leads to apoptosis. MLN4924 is currently in clinical trials. Preclinical in vivo data have indicated that MLN4924 has non-linear PK in dog and rat whole blood but not in plasma, and is extensively partitioned into red blood cells (erythrocytes, RBC). The objectives of this study were to investigate the RBC partitioning mechanism of MLN4924 in whole blood from NCR nude mice, Sprague-Dawley rats, Beagle dog, Cynomolgus monkey, and healthy human volunteers and characterize the binding site of MLN4924 in RBC. After incubation of MLN4924 at several concentrations in blood at 37°C for 60 minutes the hematocrit, and blood and plasma concentrations by LC/MS/MS were determined. The dissociation binding constant (Kd) and the maximum binding rate (Bmax) of MLN4924 to RBC were determined from the measured total and free concentrations of MLN4924 in whole blood and plasma. The RBC binding site of MLN4924 was characterized by co-incubation with carbonic anhydrase (CA) inhibitors, acetazolamide or chlorthalidone. The RBC binding of MLN4924 was found to be saturable in human, mouse, rat, dog and monkey blood and the concentration ratio of whole blood/plasma was species dependent. The Kd of MLN4924 (free concentration in blood) in the blood from human, mice, rats, dog and monkey was 0.0686, 0.0328, 0.0507, 0.0104, and 0.0126 μM, respectively, and the corresponding blood and plasma concentrations (Cb/Cp) were 55.2/2.26, 21.6/2.19, 37.2/3.87, 16.9/0.82, and 29.3/0.41 μM. The Bmax of MLN4924 in the blood from human, mice, rats, dog and monkey was 110, 43.2, 74.4, 33.8, and 58.6 μM, respectively. There was no marked difference observed in MLN4924 binding to the RBC among four individual healthy volunteers tested. The binding site of MLN4924 in the RBC was determined to be carbonic anhydrase (CA), as coincubations with CA inhibitors markedly decreased the accumulation of MLN4924 in RBCs. The binding of MLN4924 to CA in RBCs was determined to be saturable and reversible.
    No preview · Conference Paper · Aug 2010
  • Cindy Xia · Mark G. Qian · Susan Chen · Tai-Nang Huang · Frank W. Lee
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    ABSTRACT: In drug discovery, it is desirable to have a balanced tissue distribution profile for lead compounds, higher uptake into the targeted tissue (e.g., solid tumor) and low accumulation in sensitive organs (e.g., liver). Regular in-vivo pharmacokinetic studies can only provide a convoluted assessment of the property based on the parameter of the volume of distribution. To evaluate the distribution in specific tissues, individual tissue samples must be collected, homogenized, and analyzed. The processes involved are tedious, costly, and at best only suited for few final lead compounds. It is a widely accepted concept that the drug distributes into various tissues through plasma circulation. At steady state, drug in plasma reaches partition equilibrium with tissues. The free concentration in the tissue interstitial space equals to the free concentration in plasma. The total tissue concentration, however, could be different depending on the binding property of the compound with tissue proteins, membrane efflux, and uptake transporters. Based on this theory, a multiplex equilibrium dialysis device was constructed for simultaneous assessment of the drug distribution at steady state between multiple tissue homogenates and the corresponding plasma spiked with the compound of interest. This presentation will provide results that demonstrate the capability of the simple and cost-effective technique for differentiating the drug binding characteristics of different tissue homogenates against plasma proteins. Various tissues from multiple species were tested. The results generated using the novel technique are comparable to those from the in-vivo assay.
    No preview · Conference Paper · Oct 2009
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    ABSTRACT: The great emphasis on ethical and humane treatment of animals in biomedical research has culminated in the promulgation of the rule of 3Rs - Replacement, Reduction, and Refinement. We have proposed an addition to the 3Rs - a fourth R for Recycling the animal. In drug discovery single-dose pharmacokinetic studies in rats, each animal is generally used only once and then euthanized. A reduction in the number of rats used in this high-throughput era can be readily implemented by reusing animals, just as larger animals are reused in multiple pharmacokinetic studies, consequently reducing the overall number of animal lives sacrificed in research. We provide evidence here for the reproducibility of pharmaco-kinetic studies of tolbutamide and fluconazole, used as test compounds, in rats receiving once-weekly oral and intravenous doses for 4 weeks, proving that recycling rats for multiple single low-dose pharmacokinetic studies is a viable option.
    No preview · Article · Sep 2008
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    ABSTRACT: Traditional cytochrome P450 (P450) based drug-drug interaction (DDI) predictions are based on the ratio of an inhibitor's physiological concentration [I] and its inhibition constant K(i). Determining [I] at the enzymatic site, although critical for predicting clinical DDIs, remains a technical challenge. In our previous study, a novel approach using cryopreserved human hepatocytes suspended in human plasma was investigated to mimic the in vivo concentration of ketoconazole at the enzymatic site (Lu et al., 2007), effectively eliminating the estimation of the elusive [I] value. P450 inhibition in this system appears to model that in vivo. Using the ketoconazole inhibition information in a human hepatocyte-plasma suspension together with quantitative P450 phenotypic information, we successfully predicted the pharmacokinetic DDIs for a small set of drugs, such as theophylline, tolbutamide, omeprazole, desipramine, midazolam, loratadine, cyclosporine, and alprazolam, as well as an investigational compound. For the applicability of this model on a wider scale the in vitro-in vivo correlation data set needed to be expanded. However, for most drugs in the literature there is not enough quantitative information on the involvement of individual P450s to predict DDIs retrospectively. To facilitate that, in this study we determined quantitative P450 phenotyping for seven marketed drugs: budesonide, buprenorphine, loratadine, sirolimus, tacrolimus, docetaxel, and methylprednisolone. Augmentation of the new data set with the one generated previously produced broader a database that provided further support for the wider applicability of this approach using ketoconazole as a potent CYP3A inhibitor. This application is predicted to be equally effective with other P450 inhibitors that are not substrates of efflux pumps.
    No preview · Article · Aug 2008 · Drug metabolism and disposition: the biological fate of chemicals
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    ABSTRACT: Whereas ketoconazole is often used to study the worst-case scenario for clinical pharmacokinetic drug-drug interactions (DDIs) for drugs that are primarily metabolized by CYP3A4, fluconazole is considered to be a moderate inhibitor of CYP3A4, providing assessment of the moderate-case scenario of CYP3A-based DDIs. Fluconazole is also a moderate inhibitor of CYP2C9 and CYP2C19. For predicting clinical DDIs using conventional approaches, determining the in vivo inhibitor concentration at the enzymatic site [I], a critical parameter, is still not practical. In our previous study, a novel method involving hepatocyte suspension in plasma was used to circumvent the need to determine the elusive [I] value. In this study, the CYP1A2, 2C9, 2C19, 2D6, and 3A4 activities remaining in the presence of fluconazole were determined in human hepatocytes suspended in human plasma, covering a range of fluconazole clinical plasma concentrations (C(avg) and C(max)). Because the protein-binding effect of fluconazole is expected to be close to that in vivo, the inhibition observed in vitro will be similar to that in vivo. This inhibition information was then applied to the cytochrome P450 (P450) phenotypic data to predict DDIs. Using the available P450 phenotypic information on theophylline, tolbutamide, omeprazole, S-warfarin, phenytoin, cyclosporine, and midazolam and that determined in this study for sirolimus and tacrolimus, we found that the predictions for area under the curve increases for most of these drugs in the presence of fluconazole were remarkably similar (within 35%) to the observed clinical values. This study proves the general applicability of our approach using human hepatocyte incubation in human plasma to predict DDIs.
    Preview · Article · Aug 2008 · Drug metabolism and disposition: the biological fate of chemicals
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    ABSTRACT: The role of the intestine in the elimination of (2R)-2-{(3R)-3-amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide (DPC 333), a potent inhibitor of tissue necrosis factor alpha-converting enzyme, was investigated in mice and rats in vivo and in vitro. In Madine-Darby canine kidney cells stably transfected with P-glycoprotein (P-gp) and DPC 333, the transport from B-->A reservoirs exceeded the transport from A-->B by approximately 7-fold. In Caco-2 monolayers and isolated rat ileal mucosa, DPC 333 was transported from basolateral to apical reservoirs in a concentration-dependent, saturable manner, and transport was blocked by N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), confirming the contribution of P-gp/breast cancer resistance protein in B-->A efflux of DPC 333. In quantitative whole body autoradiography studies with [(14)C]DPC 333 in mice and rats, radioactivity was distributed throughout the small intestine in both species. In GF120918-pretreated bile duct-cannulated rats, radioactivity in feces was reduced 60%. Using the in situ perfused rat intestine model, approximately 20% of an i.v. dose of [(14)C]DPC 333 was measured in the intestinal lumen within 3 h postdose, 12% as parent. Kinetic analysis of data suggested that excreted DPC 333 may be further metabolized in the gut. Intestinal clearance was 0.2 to 0.35 l/h/kg. The above data suggest that in the rodent the intestine serves as an organ of DPC 333 excretion, mediated in part by the transporter P-gp.
    Preview · Article · Jun 2008 · Drug metabolism and disposition: the biological fate of chemicals
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    ABSTRACT: Low variability in plasma exposure is a desirable feature for a therapeutic agent from the aspects of efficacy and toxicity. The high plasma exposure variability is often the result of poor drug solubility, low permeability or high metabolism. It is our interest to know what is the most important factor that contributes high plasma exposure variability in clinical. Seventy-four (74) drugs were selected from the four different classes of Biopharmaceutics Classification System (BCS). Healthy volunteers’ plasma exposure data of these 74 drugs were collected from literature. There were no marked differences in Coefficient of Variation (CV, mean value of 36%) of area under plasma concentration curve (AUC) for drugs among the four different classes. Approximately 75-80% of the 74 drugs showed CV of AUC values less than 50%, while approximately 20-25% of the 74 drugs exhibited greater than 50% CV. The low CV of some low soluble and low permeable drugs could be due to the improved clinical formulation that has enhanced drug dissolution rate or permeability. Quantitative prediction of PK variability based on drug solubility, permeability, and metabolism properties remains a major challenge. Other factors such as formulation and food effect should also be considered. To delineate and estimate the contribution of major factors to exposure variation of a drug, multiple regression methods and inhibition and induction methods were exemplified by further analysis of cyclosporine and carvedilol studies from literature.
    No preview · Conference Paper · Jan 2008
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    ABSTRACT: Recently we developed a novel method for prediction of CYP3A mediated pharmacokinetic drug interactions with ketoconazole, a potent reversible inhibitor. This has enabled us to evaluate the drug interaction potential early at the preclinical stage for compounds that are cleared largely via CYP3A metabolism. The model involves measurement of inhibition of CYP activities by the compound of interest using hepatocytes suspended in neat human plasma, closely mimicking the in vivo hepatocyte conditions. The inhibition data along with CYP phenotyping data using our model predicted the AUC changes in the clinic for fifteen marketed drugs with excellent in vitro-in vivo correlationship. This approach circumvented the need of determining [I]/Ki ratio which has been marred by difficulties in determining true inhibitor concentration at the enzyme’s active site and extensive variability encountered in the determination of Ki, depending on the experimental conditions used. Though the [I]/Ki approach has provided an avenue in the past to predict the DDIs reasonably well for some compounds, more often than not the predictions were not reflective of in vivo findings. The applicability of our new model has now been successfully expanded to include a moderate CYP3A inhibitor fluconazole. The DDI predictions, retrospectively for 9 drugs and prospectively for an investigational compound, will be described in the presentation. This work further supports the validity of our model for general predictions of CYP3A mediated DDIs based on in vitro data that can be readily generated in the laboratory.
    No preview · Conference Paper · Jan 2008
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    ABSTRACT: In the discovery of new drugs for targeting solid tumors, the fast characterization of the distribution of a drug in selected tissues may be desirable for insightful information of the drugs' preferential binding to the target (tumor) and undesired distribution in susceptible organs (e.g., brains and heart muscles). Regular PK parameters, e.g., apparent volume of distribution, are indicative of only overall extravascular distribution of drugs with limited or lack of information on the nature and pattern of distribution in different tissues. Traditional distribution dialysis2 is a powerful tool but suffers from tedious and lengthy operation and effect of volume shift. This work details our efforts of using a new multiplex dialysis device for tissue binding assay with focus on shortening the incubation time, reducing the volume shift, and increasing the sample throughput and reproducibility on a more automation friendly sample format. The apparatus used was a rapid equilibrium dialysis (RED) device. The dialysis membrane has a 8,000 daltons cutoff-mass. The testing compounds were prepared in DMSO and diluted to 5 mM with blank plasma. Tissues of interest, including liver, heart, brain, kidney, lung, and tumor were homogenized in buffer. Both tissue homogenates and compound-spiked plasma were further diluted by a factor of 10 and placed in the membrane chamber (300 mL, red color coded chamber) and buffer chamber (500 mL, white color coded chamber), respectively. All experiments were conducted in triplicates unless otherwise noted. The device assembly was vortexed for 4 hours in an incubator maintained at 37 oC. At the end of dialysis, an aliquot of 50 mL samples was collected separately from both chambers. For radioactive compounds, the samples were directly ready on a Wallac Microbeta counter. The quenching factor of each compound in plasma and tissue homogenates was adjusted against the controls. For non-radioactive compounds, LC/MS/MS methods were used to measure the concentrations of the testing compounds. A protein precipitation method was used to clean up samples. Several model compounds were tested. The preliminary results indicated that the dilution of homogenate is necessary in order to reach equilibrium in a reasonable timeframe. With 5 to 10-fold dilution, the incubation time is shortened down to less than 4 hours. In addition, due to the short incubation time and the use of low cutoff mass membrane, the volume shift was reduced to a less significant level that in general doesn't require correction. The presentation will summarize the detailed results of using the device for achieving faster and reproducible tissue binding assay. 2. J. Clausen and MH Bickel, J of Pharm. Sci., 1993 (82), 345.
    No preview · Conference Paper · Jan 2008
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    ABSTRACT: Tandutinib (MLN0518) is a novel small-molecule inhibitor of FLT3, platelet-derived growth factor receptor (PDGFR), and c-kit tyrosine kinase receptors. It is being developed for the treatment of acute myeloid leukemia (AML). Tandutinib showed oral bioavailability of approximately 20% and exposure variation range from 30% to 120% (Coefficient of Variation, CV) in rats. Investigational studies in rats were conducted to explore the cause of the low plasma exposure. Tandutinib is (a) a weak base with a pH-dependent solubility profile, (b) metabolically stable in rat microsomes, and (c) a substrate of efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp). Tandutinib showed high plasma clearance with low oral bioavailability in rats. Renal clearance of tandutinib was low. In rats with a 10 mg/kg oral dose, a 5 mg/mL dosing formulation on average increased plasma exposure (area under the plasma curve, AUC) by 2-fold and reduced the Coefficient of Variation (CV) value to 40.3% compared to that of 10 mg/mL formulation (CV of 66.7%). Oral co-administration of GF120918 (an inhibitor of both P-gp and Bcrp) with tandutinib resulted in an 8-fold increase in AUC and a 3-fold reduction of CV value. P-gp inhibitors TPGS and ketoconazole increased AUC 1.5-2-fold. In fed rats, exposure to tandutinib was markedly decreased with the Cmax and AUC being approximately 8-fold and 6-fold lower, respectively, compared to fasted animals. CV values for both Cmax and AUC were doubled as well when compared to fasted animals. In summary, solubility, efflux transporters, and food all appeared to contribute to the low plasma exposure and variation of tandutinib in rats. Therefore, selection of a formulation or salt form with improved solubility, addition of efflux transporter inhibitors in the formulation, and fasting condition would be helpful in improving tandutinib oral bioavailability and exposure variability.
    No preview · Conference Paper · Jan 2008
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    ABSTRACT: Antagonism of the melanocortin 4 receptor (MC4R) has been proposed as a therapeutic intervention for the prevention of lean body mass waste, as in cachexia. Pharmacokinetic profiles of substituted 1,2-diarylethane MC4R antagonists were determined in rats after a single intravenous (IV) administration at 1 mg/kg. Brain and plasma concentrations of these compounds were determined at 1 and 4 hours after an oral dose at 10 mg/kg, since oral administration is the intended clinical dosing route and the pharmacological target is the central nervous system. The brain to plasma concentration ratios (0.10 - 50) after oral dosing correlated well with Vd(ss) (2.21 to 81.4 L/kg; R(2)=0.810) determined after IV administration. A good correlation was also observed between the brain AUC(0-4 hr) (119 - 18400 nM*hr) and Vd(ss) (R(2)=0.981). Thus, further screening and ranking of substituted 1,2-diarylethanes for their brain uptakes could be carried out more efficiently via the simple and indirect Vd(ss) screen after intravenous administration in rats.
    No preview · Article · Sep 2007
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    ABSTRACT: DPC 333 [(2R)-2-{(3R)-3-amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide] is a potent human tumor necrosis factor alpha-converting enzyme inhibitor with potential therapeutic implications for rheumatoid arthritis. Methotrexate (MTX), a drug for the treatment of rheumatoid arthritis, is eliminated primarily unchanged via renal and biliary excretion in humans as well as in rats and dogs. The objective of the present study was to investigate the potential effect of DPC 333 on the disposition of MTX. In dogs, DPC 333 administered orally at 1.7 mg/kg 15 min before the intravenous administration of [14C]MTX (0.5 mg/kg) did not alter the plasma concentration-time profile of MTX; however, the total amount of radioactivity excreted in urine increased from 58.7% to 92.2% of the dose, and the renal clearance increased from 1.8 ml/min/kg to 2.9 ml/min/kg, suggesting a decrease in MTX disposition via biliary excretion. The biliary excretion of MTX was investigated in isolated perfused livers prepared from wild-type and TR(-) [multidrug resistance-associated protein 2 (Mrp2)-deficient] Wistar rats in the absence and presence of DPC 333. Mrp2-mediated biliary excretion of MTX was confirmed with 95.8% and 5.1% of MTX recovered in the bile of wild-type and TR(-) Wistar rats, respectively. DPC 333 at an initial perfusate concentration of 50 microM completely blocked the biliary excretion of MTX, but not the clearance from perfusate, in both wild-type and TR(-) rats. These results suggest that the enhanced renal elimination of MTX may be due to the potent inhibition of biliary excretion and active renal reabsorption by DPC 333 and/or its metabolites.
    Preview · Article · Jul 2007 · Drug Metabolism and Disposition
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    ABSTRACT: The microdosing strategy allows for early assessment of human pharmacokinetics of new chemical entities using more limited safety assessment requirements than those requisite for a conventional phase I program. The current choice for evaluating microdosing is accelerator mass spectrometry (AMS) due to its ultrasensitivity for detecting radiotracers. However, the AMS technique is still expensive to be used routinely and requires the preparation of radiolabeled compounds. This report describes a feasibility study with conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology for oral microdosing assessment in rats, a commonly used preclinical species. The nonlabeled drugs fluconazole and tolbutamide were studied because of their similar pharmacokinetics characteristics in rats and humans. We demonstrate that pharmacokinetics can be readily characterized by LC-MS/MS at a microdose of 1 microg/kg for these molecules in rats, and, hence, LC-MS/MS should be adequate in human microdosing studies. The studies also exhibit linearity in exposure between the microdose and >or=1000-fold higher doses in rats for these drugs, which are known to show a linear dose-exposure relationship in the clinic, further substantiating the potential utility of LC-MS/MS in defining pharmacokinetics from the microdose of drugs. These data should increase confidence in the use of LC-MS/MS in microdose pharmacokinetics studies of new chemical entities in humans. Application of this approach is also described for an investigational compound, MLNX, in which the pharmacokinetics in rats were determined to be nonlinear, suggesting that MLNX pharmacokinetics at microdoses in humans also might not reflect those at the therapeutic doses. These preclinical studies demonstrate the potential applicability of using traditional LC-MS/MS for microdose pharmacokinetic assessment in humans.
    Preview · Article · Mar 2006 · Drug Metabolism and Disposition
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    ABSTRACT: The high-throughput screening in drug discovery for absorption, distribution, metabolism and excretion (ADME) properties has become the norm in the industry. Only a few years ago it was ADME properties that were attributed to more failure of drugs than efficacy or safety in the clinic trials. With the realization of new techniques and refinement of existing techniques better projections for the pharmacokinetic properties of compounds in humans are being made, shifting the drug failure attributes more to the safety and efficacy properties of drug candidates. There are a tremendous number of tools available to discovery scientists to screen compounds for optimization of ADME properties and selection of better candidates. However, the use of these tools has generally been to characterize these compounds rather than to select among them. This report discusses applications of the available ADME tools to better understand the clinical implication of these properties, and to optimize these properties. It also provides tracts for timing of studies with respect to the stage of the compound during discovery, by means of a discovery assay by stage (DABS) paradigm. The DABS provide the team with a rationale for the types of studies to be done during hit-to-lead, early and late lead optimization stages of discovery, as well as outlining the deliverables (objectives) at those stages. DABS has proven to be optimal for efficient utilization of resources and helped the discovery team to track the progress of compounds and projects.
    No preview · Article · Feb 2005 · Current Topics in Medicinal Chemistry
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    ABSTRACT: Single-dose pharmacokinetics of 1-aminobenzotriazole (ABT), a potent nonspecific inhibitor of cytochromes P450 (P450s), were characterized after oral administration to mice and guinea pigs at doses of 50, 100, and 150 mg/kg using serial sampling in both species. Only 30-microl blood samples were drawn from jugular vein-cannulated mice using Microvette capillary tubes containing lithium heparin. A comparison of the pharmacokinetics of antipyrine (AP) administered i.v. at 20 mg/kg to mice followed by serial and terminal sampling techniques yielded similar results. The ABT concentrations in plasma were sustained at high levels (5-100 microM) for at least 12 h in both species. Pretreatment of animals with ABT 2 h prior to AP administration decreased the plasma AP clearance by about 95% in mice at all ABT doses studied and 84, 95, and 95% in guinea pigs at a dose of 50, 100, and 150 mg/kg ABT, respectively. In vitro, the dissociation constants (KI) for ABT as the P450 mechanism-based inactivator were determined to be 45.6 and 193 microM, and the maximal inactivation rate constants (kinact) were determined to be 0.089 and 0.075 min(-1) for the mouse and guinea pig liver microsomes, respectively. The projected P450 inactivations at the plasma Cmax of ABT agreed with the inhibitions of P450-mediated AP clearance observed in vivo. For mechanistic studies in vivo overall, a 2-h prior oral pretreatment with ABT at 50 mg/kg in mice and 100 mg/kg in guinea pigs would provide significant systemic concentrations of the inhibitor over 24 h and inhibition of P450-dependent clearance of test compounds.
    No preview · Article · Nov 2004 · Drug Metabolism and Disposition
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    ABSTRACT: DPC 681 (N-[(3-fluorophenyl)methyl]glycyl-N-[3-[((3-aminophenyl) sulfonyl)-2-(aminophenyl)amino]-(1S,2S)-2-hydroxy-1-(phenyl-methyl)propyl]-3-methyl-l-valinamide) is a potent peptide-like human immunodeficiency virus protease inhibitor that was evaluated in phase I clinical trials. In primary cultures of hepatocytes, DPC 681 significantly induced the testosterone 6beta-hydroxylase activity of rat CYP3A, but not human CYP3A4. Western blot analysis, however, demonstrated a 3-fold increase in expression of CYP3A4 protein by 20 microM DPC 681 in primary cultures of human hepatocytes. Subsequent studies showed that DPC 681 was a potent inhibitor of human CYP3A4 (IC50 = 0.039 microM) and rat CYP3A (IC50 = 1.62 microM). Moreover, DPC 681 was a mechanism-based inactivator of CYP3A4 with KI and kinact of 0.24 microM and 0.22 min-1, respectively. Thus, DPC 681 is both a potent inhibitor and a strong inducer of CYP3A4. Induction of CYP3A4 by DPC 681 was masked in vitro by autoinactivation, similar to the protease inhibitor ritonavir. In pharmacokinetic studies in healthy human volunteers and rats, DPC 681 was found to highly autoinduce its metabolism. Human volunteers dosed with DPC 681 at 600 mg twice daily for 14 days had a 75% decrease in the mean area under the concentration-time curve and a more than 3-fold increase in apparent clearance as compared with that on day 1. Because the primary route of DPC 681 clearance is via CYP3A metabolism, the increased clearance observed in clinical studies is due to induction of human CYP3A4 expression.
    Full-text · Article · Oct 2003 · Drug Metabolism and Disposition

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369 Citations
73.41 Total Impact Points

Institutions

  • 2005-2011
    • Millennium Pharmaceuticals Inc
      Boston, Massachusetts, United States
  • 2002
    • Bristol-Myers Squibb
      New York City, New York, United States