Jing-Tao Wu

Strategy of utilizing in vitro and in vivo ADME tools for lead optimization and drug candidate selection.

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.

EVALUATION OF MICRODOSING TO ASSESS PHARMACOKINETIC LINEARITY IN RATS USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

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 μg/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 ≥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.

EFFECTIVE DOSING REGIMEN OF 1-AMINOBENZOTRIAZOLE FOR INHIBITION OF ANTIPYRINE CLEARANCE IN GUINEA PIGS AND MICE USING SERIAL SAMPLING

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-μl 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 μM) 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 μM, 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.

P-Glycoprotein and Breast Cancer Resistance Protein Affect Disposition of Tandutinib, A Tyrosine Kinase Inhibitor

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.

Evaluation of surrogate matrices for standard curve preparation in tissue bioanalysis

BACKGROUND The aim of this work was to investigate the feasibility of using tissue surrogate matrices and plasma for determining drug concentrations in tissues by LC-MS/MS. RESULTS The similarity of tissues was evaluated using eight model compounds with different SlogP values. For plasma dilution, the overall matrix effects of tissue samples were found to approximate to those of pure plasma samples when plasma percentage was increased. When a ten-fold plasma dilution was applied, the differences between the instrumental responses of diluted tissue samples and pure plasma samples with the same spiked concentrations were within ±20%. The results of tissue bioanalysis using plasma dilution and plasma curve for in vivo studies were acceptable with an averaged deviation of ±20-25% compared with data using traditional tissue curves. CONCLUSION Results demonstrate that tissue samples can be quantified using surrogate matrices. Plasma standard curves can be used for quantitation of both plasma and plasma-diluted tissue samples.

Online nanoelectrospray/high-field asymmetric waveform ion mobility spectrometry as a potential tool for discovery pharmaceutical bioanalysis.

Nanoelectrospray ionization (nESI) coupled online with high-field asymmetric waveform ion mobility spectrometry (FAIMS) for small molecule analysis in a discovery pharmaceutical setting was examined. A conventional capillary pump, autosampler and nESI source were used to introduce samples directly into the FAIMS device. The FAIMS device was used to separate gas-phase ions on a timescale that was compatible with the mass spectrometer. The capability of the nESI-FAIMS combination to efficiently remove metabolite interferences from the parent drug, and reduce ion suppression effects, was demonstrated. On average, 85% of the signal intensity obtained from a neat sample was preserved in the extracted plasma samples. Standard curves were prepared for several compounds. Linearity was obtained over approximately 3 to 4 orders of magnitude. Comparison of results from nESI-FAIMS with those from conventional LC/MS for a mouse pharmacokinetic study yielded concentration values differing by no more than 30%.

Case History — Use of ADME Studies for Optimization of Drug Candidates

Efficacy and safety are the two key elements in the drug discovery and development processes. The primary goal for pharmaceutical research companies is to identify and manufacture therapeutic agents that are safe and efficacious for patients. In principle, benefits versus risks have to be considered for target patient populations. The risks are relatively high in life threatening diseases, e.g. cancer, compared to general areas, e.g. inflammation. Pharmacology, medicinal chemistry, pharmaceutical sciences, safety assessment, drug metabolism and pharmacokinetics (DMPK), clinical research, etc. are the essential multidisciplinary R&D functions assembled within the pharmaceutical R&D engine to accomplish the aforementioned mission. Pharmacokinetics (PK) is generally viewed as the universal biomarker which reflects the processes of how a drug molecule is absorbed (e.g. ka), distributed (e.g. Vd) in the body, and cleared from the body through metabolism and excretion. The area under the drug plasma concentration versus time curve (AUC) provides an indirect assessment of the exposure level and duration of action of the therapeutic agent at the site of action (e.g. synovial fluid, tumor, brain). An ideal drug candidate should possess a plasma drug level which is above the therapeutic concentration (i.e. efficacious) and below the toxic concentration (i.e. safe). In general, the therapeutic index is calculated by dividing the plasma exposure at the NO (toxic) Effect Level (NOEL), or NO Adverse Effect Level (NOAEL), by the minimum plasma concentration required for efficacy (e.g. EC50) and the safety margin is calculated by dividing NOEL (or NOAEL) plasma concentration by the maximum plasma drug concentration (Cmax) achieved at an efficacious dose.

TAK-285, a Novel HER2/EGFR Inhibitor, Penetrates the CNS in Rats with an Intact Blood Brain Barrier (BBB).

Background: Approximately one third of patients with HER2-overexpressing (HER2+) metastatic breast cancer develop brain metastases. Treatment options for patients whose brain metastases have progressed after radiotherapy are urgently needed. The currently approved HER2-targeted therapies, trastuzumab and lapatinib, have generally shown limited activity against brain metastases, likely due to the limited brain penetration of these drugs. Brain penetration is mediated by multiple factors including drug permeability, tissue binding, and the expression of efflux transporters (P-gp and BCRP) and their interactions with the drug. TAK-285 is a novel, orally active, dual HER2/EGFR inhibitor currently under clinical development. In this nonclinical work, we evaluated the permeability, interactions with efflux transporters, and brain penetration of TAK-285, along with lapatinib and neratinib, using in vitro and animal models.Material and Methods: The first study evaluated the permeability of TAK-285, lapatinib, and neratinib and their interactions with efflux transporters using the Caco-2 cell monolayer system in the absence or presence of efflux pump inhibitors. The second study evaluated brain penetration of TAK-285, lapatinib and neratinib after a single oral administration in rats with an intact BBB.Results: In the Caco-2 cell model, TAK-285 showed high permeability with an efflux ratio of 1.7, indicating it is not a substrate for efflux pumps. The efflux pump inhibitors LY335979 (an inhibitor of P-gp), Ko143 (an inhibitor of BCRP), and GF120918 (an inhibitor of BCRP and P-gp) had no marked effect on the efflux ratio of TAK-285, further confirming that TAK-285 is not a substrate for P-gp and BCRP. Lapatinib and neratinib showed low to medium permeability and appeared to be substrates for P-gp/BCRP in this testing system. In the rat brain penetration study, the brain-to-plasma AUC ratio for total (free and bound) TAK-285, lapatinib, and neratinib was 0.202, 0.0243, and 0.0263, respectively.Discussion: These nonclinical studies showed that, unlike lapatinib and neratinib, TAK-285 is not a substrate for the efflux transporters, P-gp and BCRP. Based on published information, all of the approved receptor tyrosine kinase inhibitors are substrates for efflux transporters. Additionally, in rats with an intact BBB, the brain-to-plasma AUC ratio for TAK-285 was substantially higher than that of lapatinib and neratinib. The AUC ratios observed in this study for lapatinib and neratinib are similar to the proportion of blood volume in the rat brain (ie, outside the CNS) relative to the total rat brain volume. This suggests the actual brain-to-plasma AUC ratio for TAK-285 in rats may be much greater than 8-fold of that of lapatinib and neratinib calculated based on these measured AUC ratios. Consequently, TAK-285 may have a unique role in the treatment of brain lesions in patients with HER2- or EGFR-dependent tumors. Furthermore, TAK-285 may not be subject to drug resistance conferred by efflux transporters. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5098.

The use of a rapid MS-based method for the quantification of the CYP 3A4 protein directly from hepatocyte cell lysate for CYP induction studies

BACKGROUND Most P450 protein quantitation methods involved the time-consuming preparation of microsomes and therefore are not amenable for high-throughput analysis. We here report a new method to measure P450 CYP3A4 protein levels directly from cell lysates. RESULTS A direct sample preparation method from hepatocyte cell lysate has been developed for the quantification of CYP3A4 protein levels by combining a modified semi-automated precipitation with a filter-aided sample preparation. This novel LC-MS/MS-based method provides simple, subfemtomole sensitivity and rapid quantitation of CYP3A4 protein levels directly from hepatocyte lysate without the need for microsome preparation. CONCLUSION A rapid, accurate and sensitive method has been developed and implemented to quantify CYP3A4 protein in hepatocytes down to 0.05 million cells in CYP induction studies. The number of cells required for quantitation was well below the typical 0.25 million cells used in a CYP induction study.

An Indirect Screen for Brain Uptake of 1,2-Diarylethane Melanocortin 4 Receptor Antagonists in Rats

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.