Multicenter Validation Study of Quantitative Imaging Mass Spectrometry.

The aim of this study was to assess potential sources of variability in quantitative imaging mass spectrometry (IMS) across multiple sites, analysts, and instruments. A sample from rat liver perfused with clozapine was distributed to three sites for analysis by three analysts using a predefined protocol to standardize the sample preparation, acquisition, and data analysis parameters. In addition, two commonly used approaches to IMS quantification, the mimetic tissue model and dilution series, were used to quantify clozapine and its major metabolite norclozapine in isolated perfused rat liver. The quantification was evaluated in terms of precision and accuracy with comparison to liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The results of this study showed that, across three analysts with six replicates each, both quantitative IMS methods achieved relative standard deviations in the low teens and accuracies of around 80% compared to LC-MS/MS quantification of adjacent tissue sections. The utility of a homogeneously coated stable-isotopically labeled standard (SIL) for normalization was appraised in terms of its potential to improve precision and accuracy of quantification as well as qualitatively reduce variability in the sample tissue images. SIL normalization had a larger influence on the dilution series, where the use of the internal standard was necessary to achieve accuracy and precision comparable to the non-normalized mimetic tissue model data. Normalization to the internal standard appeared most effective when the intensity ratio of the analyte to internal standard was approximately one, and thus precludes this method as a universal normalization approach for all ions in the acquisition.

An Investigation into Retigabine (Ezogabine) Associated Dyspigmentation in Rat Eyes by MALDI Imaging Mass Spectrometry.

Retigabine (RTG) is an antiepileptic drug approved as an adjunctive treatment for refractory partial-onset seizures in adults. In April 2013, the Food and Drug Administration issued a warning that RTG could cause changes in retinal pigmentation and discoloration of skin, resulting in a blue appearance. As part of a larger preclinical effort to gain a mechanistic understanding as to the origins of retinal pigment changes associated with RTG, we conducted a long-term repeat dosing study in rats. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) was used to determine the distribution of RTG and its metabolites in the rat eye following 13 and 39 weeks of dosing. IMS revealed the presence of RTG, a previously characterized N-acetyl metabolite of RTG (NAMR), and several species structurally related through the dimerization of RTG and NAMR. These species were highly localized to the melanin-containing layers of the uveal tract of the rat eye including the choroid, ciliary body, and iris, suggesting that the formation of these dimers occurs from melanin bound RTG and NAMR. Furthermore, several of the RTG-related dimers have UV absorbance which give them a purple color in solution. We propose that the melanin binding of RTG and NAMR effectively concentrates the two compounds to enable mixed condensation reactions to occur when the binding provides the proper geometry in the redox environment of the uveal tissues. High lateral resolution images illustrate that the blood-retinal barrier effectively restricts retinal access to RTG-related compounds. The spatial information provided by MALDI IMS was critical in contextualizing the homogenate concentrations of key RTG-related compounds and helped provide a basis for the mechanism of dimer formation.

Drug-induced Liver Fibrosis: Testing Nevirapine in a Viral-like Liver Setting Using Histopathology, MALDI IMS, and Gene Expression.

Nevirapine (NVP) is associated with hepatotoxicity in 1-5% of patients. In rodent studies, NVP has been shown to cause hepatic enzyme induction, centrilobular hypertrophy, and skin rash in various rat strains but not liver toxicity. In an effort to understand whether NVP is metabolized differently in a transiently inflamed liver and whether a heightened immune response alters NVP-induced hepatic responses, female brown Norway rats were dosed with either vehicle or NVP alone (75 mg/kg/day for 15 days) or galactosamine alone (single intraperitoneal [ip] injection on day 7 to mimic viral hepatitis) or a combination of NVP (75/100/150 mg/kg/day for 15 days) and galactosamine (single 750 mg/kg ip on day 7). Livers were collected at necropsy for histopathology, matrix-assisted laser desorption/ionization imaging mass spectrometry and gene expression. Eight days after galactosamine, hepatic fibrosis was noted in rats dosed with the combination of NVP and galactosamine. No fibrosis occurred with NVP alone or galactosamine alone. Gene expression data suggested a viral-like response initiated by galactosamine via RNA sensors leading to apoptosis, toll-like receptor, and dendritic cell responses. These were exacerbated by NVP-induced growth factor, retinol, apoptosis, and periostin effects. This finding supports clinical reports warning against exacerbation of fibrosis by NVP in patients with hepatitis C.

Assessing drug and metabolite detection in liver tissue by UV-MALDI and IR-MALDESI mass spectrometry imaging coupled to FT-ICR MS.

Determining the distribution of a drug and its metabolites within tissue is a key facet of evaluating drug candidates. Drug distribution can have a significant implication in appraising drug efficacy and potential toxicity. The specificity and sensitivity of mass spectrometry imaging (MSI) make it a perfect complement to the analysis of drug distributions in tissue. The detection of lapatinib as well as several of its metabolites in liver tissue was determined by MSI using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to high resolving power Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. IR-MALDESI required minimal sample preparation while maintaining high sensitivity. The effect of the electrospray solvent composition on IR-MALDESI MSI signal from tissue analysis was investigated and an empirical comparison of IR-MALDESI and UV-MALDI for MSI analysis is also presented.

Effects of enzyme inducers efavirenz and tipranavir/ritonavir on the pharmacokinetics of the HIV integrase inhibitor dolutegravir.

PURPOSE: Dolutegravir (DTG) is an unboosted, integrase inhibitor for the treatment of HIV infection. Two studies evaluated the effects of efavirenz (EFV) and tipranavir/ritonavir (TPV/r) on DTG pharmacokinetics (PK) in healthy subjects. METHODS: The first study was an open-label crossover where 12 subjects received DTG 50 mg every 24 hours (q24h) for 5 days, followed by DTG 50 mg and EFV 600 mg q24h for 14 days. The second study was an open-label crossover where 18 subjects received DTG 50 mg q24h for 5 days followed by TPV/r 500/200 mg every 12 hours (q12h) for 7 days and then DTG 50 mg q24h and TPV/r 500/200 mg q12h for a further 5 days. Safety assessments and serial PK samples were collected. Non-compartmental PK analysis and geometric mean ratios and 90% confidence intervals were generated. RESULTS: The combination of DTG with EFV or TPV/r was generally well tolerated. Four subjects discontinued the TPV/r study due to increases in alanine aminotransferase that were considered related to TPV/r. Co-administration with EFV resulted in decreases of 57, 39 and 75% in DTG AUC(0-τ), Cmax and Cτ, respectively. Co-administration with TPV/r resulted in decreases of 59, 46 and 76% in DTG AUC(0-τ), Cmax and Cτ, respectively. CONCLUSIONS: Given the reductions in exposure and PK/pharmacodynamic relationships in phase II/III trials, DTG should be given at an increased dose of 50 mg twice daily when co-administered with EFV or TPV/r, and alternative regimens without inducers should be considered in integrase inhibitor-resistant patients.

Cardiolipin profiles as a potential biomarker of mitochondrial health in diet-induced obese mice subjected to exercise, diet-restriction and ephedrine treatment.

Cardiolipin (CL) is crucial for mitochondrial energy metabolism and structural integrity. Alterations in CL quantity or CL species have been associated with mitochondrial dysfunction in several pathological conditions and diseases, including mitochondrial dysfunction-related compound attrition and post-market withdrawal of promising drugs. Here we report alterations in the CL profiles in conjunction with morphology of soleus muscle (SM) and brown adipose tissue (BAT) in diet-induced obese (DIO) mice, subjected to ephedrine treatment (EPH: 200 mg kg(-1) day(-1) orally), treadmill exercise (EX: 10 meters per min, 1 h per day), or dietary restriction (DR: 25% less of mean food consumed by the EX group) for 7 days. Mice from the DR and EPH groups had a significant decrease in percent body weight and reduced fat mass compared with DIO controls. Morphologic alterations in the BAT included brown adipocytes with reduced cytoplasmic lipid droplets and increased cytoplasmic eosinophilia in the EX, DR and EPH groups. Increased cytoplasmic eosinophilia in the BAT was ultrastructurally manifested by increased mitochondrial cristae, fenestration of mitochondrial cristae, increased electron density of mitochondrial matrix, and increased complexity of shape and elongation of mitochondria. Mitochondrial ultrastructural alterations in the SM of the EX and DR groups included increased mitochondrial cristae, cup-shaped mitochondria and mitochondrial degeneration. All four CL species (tri-linoleoyl-mono-docosahexaenoyl, tetralinoleoyl, tri-linoleoyl-mono-oleoyl, and di-linoleoyl-di-oleoyl) were increased in the BAT of the DR and EPH groups and in the SM of the EPH and EX groups. In conclusion, cardiolipin profiling supported standard methods for assessing mitochondrial biogenesis and health, and may serve as a potential marker of mitochondrial dysfunction in preclinical toxicity studies.

Metabolism, excretion, and mass balance of the HIV-1 integrase inhibitor dolutegravir in humans.

The pharmacokinetics, metabolism, and excretion of dolutegravir, an unboosted, once-daily human immunodeficiency virus type 1 integrase inhibitor, were studied in healthy male subjects following single oral administration of [(14)C]dolutegravir at a dose of 20 mg (80 µCi). Dolutegravir was well tolerated, and absorption of dolutegravir from the suspension formulation was rapid (median time to peak concentration, 0.5 h), declining in a biphasic fashion. Dolutegravir and the radioactivity had similar terminal plasma half-lives (t1/2) (15.6 versus 15.7 h), indicating metabolism was formation rate limited with no long-lived metabolites. Only minimal association with blood cellular components was noted with systemic radioactivity. Recovery was essentially complete (mean, 95.6%), with 64.0% and 31.6% of the dose recovered in feces and urine, respectively. Unchanged dolutegravir was the predominant circulating radioactive component in plasma and was consistent with minimal presystemic clearance. Dolutegravir was extensively metabolized. An inactive ether glucuronide, formed primarily via UGT1A1, was the principal biotransformation product at 18.9% of the dose excreted in urine and the principal metabolite in plasma. Two minor biotransformation pathways were oxidation by CYP3A4 (7.9% of the dose) and an oxidative defluorination and glutathione substitution (1.8% of the dose). No disproportionate human metabolites were observed.

A mimetic tissue model for the quantification of drug distributions by MALDI imaging mass spectrometry.

The full potential of imaging mass spectrometry (IMS) as a tool in drug development will not be realized until reliable quantitative information can be integrated with the molecular distributions. Here we report a novel method for the quantification of drugs in tissue sections using matrix-assisted laser desorption/ionization (MALDI) IMS. This method uses a mimetic tissue model consisting of a set of tissue homogenates spiked with a range of different drug concentrations that have been frozen into a polymer support mold. The goal of this model is to mimic a dosed tissue in its effects on analyte extraction and ion suppression. Parallel preparation and analysis of sections from the tissue model and the dosed tissues allow for the quantification of a drug's distribution. Here we detail the steps involved in constructing the model and provide proof of concept data to highlight the potential of this approach. Several figures of merit are evaluated including linearity of response, variability, and section-to-section reproducibility. Finally, the tissue model is used to quantify two different drugs, lapatinib and nevirapine, in dosed tissues from nonclinical species and the results are compared with those generated by LC-MS quantification.

Central nervous system disposition and metabolism of Fosdevirine (GSK2248761), a non-nucleoside reverse transcriptase inhibitor: an LC-MS and Matrix-assisted laser desorption/ionization imaging MS investigation into central nervous system toxicity.

The CNS disposition and metabolism of Fosdevirine (FDV), an HIV non-nucleoside reverse transcriptase inhibitor, was investigated in four patients who unexpectedly experienced seizures after at least 4 weeks of treatment in a Phase IIb, HIV-1 treatment experienced study. In addition, the CNS disposition and metabolism of FDV was examined in samples from rabbit, minipig, and monkey studies. LC-MS was used to characterize and estimate the concentrations of FDV and its metabolites in cerebral spinal fluid (seizure patients, rabbit, and monkey) and brain homogenate (rabbit, minipig, and monkey). The application of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) provided the spatial distribution of FDV and its metabolites in brain tissue (rabbit, minipig, and monkey). A cysteine conjugate metabolite resulting from an initial glutathione (GSH) Michael addition to the trans-phenyl acrylonitrile moiety of FDV was the predominant drug-related component in the samples from seizure patients, rabbits, and minipigs. This metabolite persisted in the CNS for an extended period of time after the last dose in both seizure patients and minipigs. Furthermore, the localization of this metabolite was found to be highly associated with the white matter in rabbit and minipig brain sections by MALDI IMS. In contrast, the predominant component in monkey CNS was FDV, which was shown to be highly associated with the gray matter. On the basis of these data, several hypothesizes are considered, which might provide insights into species differences in CNS toxicity/seizures observed after FDV dosing.

Human metabolism of lapatinib, a dual kinase inhibitor: implications for hepatotoxicity.

Lapatinib (Tykerb, Tyverb) is an important orally active dual tyrosine kinase inhibitor efficacious in combination therapy for patients with progressive human epidermal receptor 2-overexpressing metastatic breast cancer. However, clinically significant liver injury, which may be associated with lapatinib metabolic activation, has been reported. We describe the metabolism and excretion of [(14)C]lapatinib in six healthy human volunteers after a single oral dose of 250 mg and the potential relationships between metabolism and clinical hepatotoxicity. Overall, elimination showed high intersubject variability, with fecal elimination being the predominant pathway, representing a median of 92% of the dose with lapatinib as the largest component (approximate median 27% of the dose). In plasma, approximately 50% of the observed radioactivity was attributed to metabolites. Analysis of a 4-h pooled plasma extract identified seven metabolites related by an N- and α-carbon oxidation cascade. Fecal metabolites derived from three prominent pathways: N- and α-carbon oxidation, fluorobenzyl oxidative cleavage, and hydroxypyridine formation. Several of the lapatinib metabolites can undoubtedly be linked to reactive species such as aldehydes or quinone imines. In addition to the contribution of these potentially reactive metabolites as suspects in clinical liver injury, the role of other disposition factors, including interaction with drug transporters, pharmacogenetics, or magnitude of the therapeutic dose, should not be discounted.

Assessment of the drug interaction risk for remogliflozin etabonate, a sodium-dependent glucose cotransporter-2 inhibitor: evidence from in vitro, human mass balance, and ketoconazole interaction studies.

Remogliflozin etabonate is the ester prodrug of remogliflozin, a selective sodium-dependent glucose cotransporter-2 inhibitor. This work investigated the absorption, metabolism, and excretion of [(14)C]remogliflozin etabonate in humans, as well as the influence of P-glycoprotein (Pgp) and cytochrome P450 (P450) enzymes on the disposition of remogliflozin etabonate and its metabolites to understand the risks for drug interactions. After a single oral 402 ± 1.0 mg (106 ± 0.3 µCi) dose, [(14)C]remogliflozin etabonate is rapidly absorbed and extensively metabolized. The area under the concentration-time curve from 0 to infinity [AUC((0-∞))] of plasma radioactivity was approximately 14-fold higher than the sum of the AUC((0-∞)) of remogliflozin etabonate, remogliflozin, and 5-methyl-4-({4-[(1-methylethyl)oxy]phenyl}methyl)-1H-pyrazol-3-yl-ß-d-glucopyranoside (GSK279782), a pharmacologically active N-dealkylated metabolite. Elimination half-lives of total radioactivity, remogliflozin etabonate, and remogliflozin were 6.57, 0.39, and 1.57 h, respectively. Products of remogliflozin etabonate metabolism are eliminated primarily via renal excretion, with 92.8% of the dose recovered in the urine. Three glucuronide metabolites made up the majority of the radioactivity in plasma and represent 67.1% of the dose in urine, with 5-methyl-1-(1-methylethyl)-4-({4-[(1-methylethyl)oxy]phenyl}methyl)-1H-pyrazol-3-yl-ß-d-glucopyranosiduronic acid (GSK1997711) representing 47.8% of the dose. In vitro studies demonstrated that remogliflozin etabonate and remogliflozin are Pgp substrates, and that CYP3A4 can form GSK279782 directly from remogliflozin. A ketoconazole clinical drug interaction study, along with the human mass balance findings, confirmed that CYP3A4 contributes less than 50% to remogliflozin metabolism, demonstrating that other enzyme pathways (e.g., P450s, UDP-glucuronosyltransferases, and glucosidases) make significant contributions to the drug's clearance. Overall, these studies support a low clinical drug interaction risk for remogliflozin etabonate due to the availability of multiple biotransformation pathways.

Lapatinib distribution in HER2 overexpressing experimental brain metastases of breast cancer.

PURPOSE: Lapatinib, a small molecule EGFR/HER2 inhibitor, partially inhibits the outgrowth of HER2+ brain metastases in preclinical models and in a subset of CNS lesions in clinical trials of HER2+ breast cancer. We investigated the ability of lapatinib to reach therapeutic concentrations in the CNS following (14)C-lapatinib administration (100 mg/kg p.o. or 10 mg/kg, i.v.) to mice with MDA-MD-231-BR-HER2 brain metastases of breast cancer. METHODS: Drug concentrations were determined at differing times after administration by quantitative autoradiography and chromatography. RESULTS: (14)C-Lapatinib concentration varied among brain metastases and correlated with altered blood-tumor barrier permeability. On average, brain metastasis concentration was 7-9-fold greater than surrounding brain tissue at 2 and 12 h after oral administration. However, average lapatinib concentration in brain metastases was still only 10-20% of those in peripheral metastases. Only in a subset of brain lesions (17%) did lapatinib concentration approach that of systemic metastases. No evidence was found of lapatinib resistance in tumor cells cultured ex vivo from treated brains. CONCLUSIONS: Results show that lapatinib distribution to brain metastases of breast cancer is partially restricted and blood-tumor barrier permeability is a key component of lapatinib therapeutic efficacy which varies between tumors.

Mapping the Lipids of Skin Sebaceous Glands and Hair Follicles by High Spatial Resolution MALDI Imaging Mass Spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a technology that utilizes the high sensitivity and specificity of mass spectrometry, combined with a high spatial resolution to characterize the molecular species present in skin tissue. In this article, we use MALDI IMS to map specific lipids characteristic of two important skin appendages in minipig skin: the sebaceous glands and hair follicles. A set of specific lipid markers linked to the synthesis of sebum, stages of sebum production, and the secretion of sebum for two different sebaceous gland subzones, the peripheral and central necrotic, were identified. Furthermore, biochemical pathway analysis of the identified markers provides potential drug-targeting strategies to reduce sebum overproduction in pathological conditions. In addition, specific lipid markers characteristic of the different layers in the hair follicle bulge area, including the outer root sheath, the inner root sheath, and the medulla that are associated with the growth cycles of the hair, were determined. This research highlights the ability of MALDI IMS to link a molecular distribution not only to the morphological features in skin tissue but to the physiological state as well. Thus, this platform can provide a basis for the investigation of biochemical pathways as well as the mechanisms of disease and pharmacology in the skin, which will ultimately be critical for drug discovery and the development of dermatology-related illnesses.

GlycoFibroTyper: A Novel Method for the Glycan Analysis of IgG and the Development of a Biomarker Signature of Liver Fibrosis.

Our group has recently developed the GlycoTyper assay which is a streamlined antibody capture slide array approach to directly profile N-glycans of captured serum glycoproteins including immunoglobulin G (IgG). This method needs only a few microliters of serum and utilizes a simplified processing protocol that requires no purification or sugar modifications prior to analysis. In this method, antibody captured glycoproteins are treated with peptide N-glycosidase F (PNGase F) to release N-glycans for detection by MALDI imaging mass spectrometry (IMS). As alterations in N-linked glycans have been reported for IgG from large patient cohorts with fibrosis and cirrhosis, we utilized this novel method to examine the glycosylation of total IgG, as well as IgG1, IgG2, IgG3 and IgG4, which have never been examined before, in a cohort of 106 patients with biopsy confirmed liver fibrosis. Patients were classified as either having no evidence of fibrosis (41 patients with no liver disease or stage 0 fibrosis), early stage fibrosis (10 METAVIR stage 1 and 18 METAVIR stage 2) or late stage fibrosis (6 patients with METAVIR stage 3 fibrosis and 37 patients with METAVIR stage 4 fibrosis (cirrhosis)). Several major alterations in glycosylation were observed that classify patients as having no fibrosis (sensitivity of 92% and a specificity of 90%), early fibrosis (sensitivity of 84% with 90% specificity) or significant fibrosis (sensitivity of 94% with 90% specificity).

Multimodal imaging distribution assessment of a liposomal antibiotic in an infectious disease model.

Liposomes are promising targeted drug delivery systems with the potential to improve the efficacy and safety profile of certain classes of drugs. Though attractive, there are unique analytical challenges associated with the development of liposomal drugs including human dose prediction given these are multi-component drug delivery systems. In this study, we developed a multimodal imaging approach to provide a comprehensive distribution assessment for an antibacterial drug, GSK2485680, delivered as a liposomal formulation (Lipo680) in a mouse thigh model of bacterial infection to support human dose prediction. Positron emission tomography (PET) imaging was used to track the in vivo biodistribution of Lipo680 over 48 h post-injection providing a clear assessment of the uptake in various tissues and, importantly, the selective accumulation at the site of infection. In addition, a pharmacokinetic model was created to evaluate the kinetics of Lipo680 in different tissues. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was then used to quantify the distribution of GSK2485680 and to qualitatively assess the distribution of a liposomal lipid throughout sections of infected and non-infected hindlimb tissues at high spatial resolution. Through the combination of both PET and MALDI IMS, we observed excellent correlation between the Lipo680-radionuclide signal detected by PET with the GSK2485680 and lipid component signals detected by MALDI IMS. This multimodal translational method can reduce drug attrition by generating comprehensive biodistribution profiles of drug delivery systems to provide mechanistic insight and elucidate safety concerns. Liposomal formulations have potential to deliver therapeutics across a broad array of different indications, and this work serves as a template to aid in delivering future liposomal drugs to the clinic.

The emergence of imaging mass spectrometry in drug discovery and development: Making a difference by driving decision making.

The pharmaceutical industry is a dynamic, science-driven business constantly under pressure to innovate and morph into a higher performing organization. Innovations can include the implementation of new technologies, adopting new scientific methods, changing the decision-making process, compressing timelines, or making changes to the organizational structure. The drivers for the constant focus on performance improvement are the high cost of R&D as well as the lengthy timelines required to deliver new medicines for unmet needs. Successful innovations are measured against both the quality and quantity of potential new medicines in the pipeline and the delivery to patients. In this special feature article, we share our collective experience implementing matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) technology as an innovative approach to better understand the tissue biodistribution of drugs in the early phases of drug discovery to establish pharmacokinetic-pharmacodynamic (PK-PD) relationships, as well as in the development phase to understand pharmacology, toxicology, and disease pathogenesis. In our experience, successful implementation of MALDI IMS in support of therapeutic programs can be measured by the impact IMS studies have on driving decision making in pipeline progression. This provides a direct quantifiable measurement of the return to the organization for the investment in IMS. We have included discussion not only on the technical merits of IMS study conduct but also the key elements of setting study objectives, building collaborations, data integration into the medicine progression milestones, and potential pitfalls when trying to establish IMS in the pharmaceutical arena. We categorized IMS study types into five groups that parallel pipeline progression from the earliest phases of discovery to late stages of preclinical development. We conclude the article with some perspectives on how we see MALDI IMS maintaining relevance and becoming further embedded as an essential tool in the constantly changing environment of the pharmaceutical industry.

The disposition and metabolism of GW695634: a non-nucleoside reverse transcriptase inhibitor (NNRTi) for treatment of HIV/AIDS.

GW695634 is the prodrug of GW678248, a novel non-nucleoside reverse transcriptase inhibitor with potent antiviral activity against HIV/AIDS efavirenz- and nevirapine-resistant viruses. In mice, rats, and monkeys following oral administration of [(14)C]GW695634, the primary pathway of metabolic clearance was by amide hydrolysis and the main route of elimination (46%-75% of the dose) was in the feces. The primary metabolic pathway of clearance for GW695634 and GW678248 in the preclinical species was by amide hydrolysis. At least six metabolites were observed that were the products of GW695634 and GW678248 amide hydrolysis.

An unexpected synergist role of P-glycoprotein and breast cancer resistance protein on the central nervous system penetration of the tyrosine kinase inhibitor lapatinib (N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine; GW572016).

Lapatinib is a tyrosine kinase inhibitor approved for use in combination with capecitabine to treat advanced or metastatic breast cancers overexpressing human epidermal receptor 2 (ErbB2). This work investigated the role of P-glycoprotein (Pgp; the protein from the Mdr1a/b gene) and breast cancer resistance protein (Bcrp; the protein from the Bcrp1 gene) in modulating the central nervous system penetration of lapatinib at steady-state conditions in FVBn mice (wild-type), Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)/Bcrp1(-/-) knockout mice. After an intravenous infusion of lapatinib for 24 h to a targeted steady-state plasma concentration of 700 ng/ml (0.3 mg/kg/h) or 7000 ng/ml (3 mg/kg/h), lapatinib brain-to-plasma ratios were approximately 3- to 4-fold higher in Mdr1a/b(-/-) knockout mice (ratio range from 0.09 to 0.16) compared with wild-type mice (ratio range from 0.03 to 0.04). There was no difference in the brain-to-plasma ratio in the Bcrp1(-/-) knockout mice (ratio range from 0.03 to 0.04) compared with wild-type mice. In contrast, Mdr1a/b(-/-)/Bcrp1(-/-) triple knockout mice had a 40-fold higher brain-to-plasma ratio (ratio range from 1.2 to 1.7), suggesting that Pgp and Bcrp work in concert to limit the brain-to-plasma ratio of lapatinib in mice. This finding has important potential consequences for the treatment of brain tumors in breast cancer patients treated with tyrosine kinase inhibitors as well as the basic understanding of ATP binding cassette transporters expressed in the blood-brain barrier on the central nervous system disposition of drugs.

Quantification and assessment of detection capability in imaging mass spectrometry using a revised mimetic tissue model.

Aim: A revised method of preparing the mimetic tissue model for quantitative imaging mass spectrometry (IMS) is evaluated. Concepts of assessing detection capability are adapted from other imaging or mass spectrometry (MS)-based technologies to improve upon the reliability of IMS quantification. Materials & methods: The mimetic tissue model is prepared by serially freezing spiked-tissue homogenates into a cylindrical mold to create a plug of tissue with a stepped concentration gradient of matrix-matched standards. Weighted least squares (WLS) linear regression is applied due to the heteroscedastisity (change in variance with intensity) of most MS data. Results & conclusions: Imaging poses several caveats for quantification which are unique compared with other MS-based methods. Aspects of the design, construction, application, and evaluation of the matrix-matched standard curve for the mimetic tissue model are discussed. In addition, the criticality of the ion distribution in the design of a purposeful liquid chromatography coupled to mass spectrometry (LC-MS) validation is reviewed.

The role of efflux and uptake transporters in [N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (GW572016, lapatinib) disposition and drug interactions.

Lapatinib [N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine, GW572016, Tykerb] is a tyrosine kinase inhibitor approved for use in combination with capecitabine to treat advanced or metastatic breast cancers overexpressing HER2 (ErbB2). In this work we investigated the role of efflux and uptake transporters in lapatinib disposition and drug interactions. In vitro studies evaluated whether lapatinib is a substrate for efflux transporters or an inhibitor of efflux/uptake transporters. In vivo studies included whole-body autoradiography and an evaluation of the role of efflux transporters on the intestinal absorption and brain penetration of lapatinib using chemical or genetic knockout animals. Lapatinib is a substrate for the efflux transporters P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP). Furthermore, lapatinib is an inhibitor (IC(50) values 0.025-5 muM) of Pgp, BCRP, and organic anion transporting polypeptide 1B1 (a hepatic uptake transporter). In contrast, lapatinib yielded little inhibition on renal transporters (organic anion transporters, organic cation transporters, and uric acid transporter). In vivo studies demonstrated that brain concentrations of lapatinib were low and influenced by efflux transporters at the blood-brain barrier. In contrast, systemic exposure of lapatinib after oral dosing was unchanged when efflux by Pgp and BCRP was absent from the gastrointestinal tract. These in vitro and in vivo preclinical investigations provide a mechanistic basis for elucidating clinical drug interactions.