Article pubs.acs.org/crt
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 Stephen Castellino,*,† M. Reid Groseclose,† James Sigafoos,† David Wagner,† Mark de Serres,† Joseph W. Polli,† Elizabeth Romach,‡ James Myer,‡ and Brad Hamilton‡ †
Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, United States Safety Assessment, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, United States
‡
S Supporting Information *
ABSTRACT: 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 matrixassisted 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.
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INTRODUCTION
decreases in HIV-1 plasma RNA from baseline to day 8 up to 1.83 log10 at doses of 100 to 800 mg (QD). However, in a phase IIb clinical trial (SGN113399; US Clinical Trials Identifier NCT01199731) in treatment-experienced HIV-infected patients, 5 subjects of 20 enrolled in the treatment arms of FDV (100 and 200 mg, QD), in combination with raltegravir and ritonavir boosted darunavir, experienced seizures after the completion of at least 4 weeks of treatment. The high incidence of seizures observed in the SGN113399 trial could not be explained by background seizure incidence or seizure risk of other concomitant medications. One subject had a family history of seizures (mother). Two subjects had preexisting brain pathology of unknown significance (agenesis of the corpus collosu and asymmetric cerebral atrophy) (Margolis, D., et al., unpublished results). Prior to the phase IIb study, 187
Fosdevirine (FDV), GSK2248761 (IDX899), is a potent, selective, non-nucleoside reverse transcriptase inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1) replication with low nanomolar activity in vitro. FDV shows good activity against a broad range of HIV-1 strains, including efavirenz (EFV)-resistant clinical isolates. Resistance to FDV develops slower than to EFV in vitro.1,2 Additionally, FDV exhibited potent in vitro antiviral efficacy against K103N and Y181C single point mutations and was equally effective against most double mutants.3−5 In phase I single-dose escalation (50 to 1,200 mg) and 7-day multiple-dose (800 mg QD or 400 mg BID) studies in healthy male subjects, FDV was well tolerated with no serious adverse events or laboratory abnormalities detected.6 In a 7-day randomized, sequential cohort, doubleblind, placebo-controlled proof-of concept study in treatmenẗ HIV-1-infected subjects, FDV was well tolerated and naive demonstrated potent HIV-1 antiviral activity with mean © 2012 American Chemical Society
Received: October 9, 2012 Published: December 10, 2012 241
dx.doi.org/10.1021/tx3004196 | Chem. Res. Toxicol. 2013, 26, 241−251
Chemical Research in Toxicology
Article
mol) with human liver microsomes. FDV, the isolated metabolites, and plasma obtained from cynomolgus monkey dosed with [14C]FDV (sp act 1.37 μCi/mol) were used as reference standards for estimating relative amounts of FDV-related material in biological samples. All samples and extracts were stored at −20 °C or below when not in use. All other chemicals used during this study were reagent grade or better and were obtained from standard chemical suppliers. Sample Collection. All human and animal studies were conducted in approved facilities after approval of the protocols and patient informed consent by an Investigational Review Board Ethics Committee or Institutional Animal Care and Use Committees as appropriate. Human Samples. A single CSF sample was obtained from four HIV-infected patients who experience seizures at 6, 18, 22, or 40 days postdose following completion of repeat oral dosing at 100 or 200 mg. The samples were heat-inactivated by incubation for five hours at 60 °C in a shaking water bath. The recovery of FDV spiked into control human CSF (BioRecalmation, LLC, Hicksville, NY) was calculated to be 86% following heat treatment by comparing the peak areas of the FDV mass spectral extracted ion chromatograms of heated and unheated samples. Rabbit Samples. One female Dutch Belted rabbit was administered repeat oral gavage dosing of FDV (125 mg/kg/day) for 7 days. Rabbit CSF, plasma, and brain tissue were collected 3 h after the final dose. The brain was divided along the midline and flash frozen in liquid nitrogen at the time of necropsy. Monkey Samples. One male cynomolgus monkey (Macaca fasicularis; Charles River Laboratories BRF, Houston TX) was administered repeat oral gavage dosing of FDV (200 mg/kg/day) for 7 days. Monkey CSF, plasma, and brain tissue were collected 3 h after the final dose. At the time of necropsy, the brain was divided at the midline, and coronally at the level of the frontal lobes/anterior brain (section 1), the optic chiasm (section 2), the pituitary (section 3), the pons (section 4), and the cerebellum/brainstem (section 5). The brain sections were flash frozen in liquid nitrogen immediately after sectioning. Minipig Samples. Minipigs were administered single oral gavage doses of 100, 500, and 1,000 mg/kg FDV, with a 4 day washout period between each dose escalation. Brains were collected at 21 days (animal #20) or 25 days (animal#17) following the 1,000 mg/kg dose. The brains were sectioned at the midline and flash frozen in liquid nitrogen at the time of collection. Metabolite Estimations. FDV and radiolabeled biological reference isolates of M6, M16, M22, M42, and FDV were spiked into control CSF, plasma, and brain homogenate extracts to facilitate estimation of metabolites in the samples with various matrices. In addition, plasma extracts from cynomolgus monkeys dosed with [14C]FDV (25 mg/kg) assisted the estimation of DRM (drug-related material) in the samples. These biological reference standards were analyzed by LC-MS/MS at the time of sample analysis. Fractions from the postcolumn split were dried under nitrogen and counted on a Topcount NXT microscintillation plate counter after the addition of Microscint PS scintillation cocktail. Data were analyzed using Laura software (v3.4.11.64) to determine the amount of radioactivity in each peak of interest. Relative MS response factors (area counts/pmol standard) were generated by correlating the MS response from extracted ion chromatograms of the molecular ions to the amount calculated by radioactivity. The relative MS response factor for each standard was used to estimate the amount of each metabolite in the samples. In instances where biological standards were not available, MS response factors for structurally similar metabolites or FDV were used in calculations. Imaging Mass Spectrometry. Sagittal sections (12 μm) of frozen brain were collected in a cryostat and mounted onto indium tin oxide coated glass microscope slides (Bruker Daltonics, Billerica, MA). Sections serial to those collected for imaging mass spectrometry (IMS) were collected and stained with hematoxylin and eosin (H&E) for histological analysis and correlation to IMS. Additional sections were also collected for LC-MS analysis to enable the estimation of metabolite quantities.
human subjects had been exposed to FDV in the clinic without a CNS incident. The standard package of preclinical toxicity data available prior to the phase IIb study did not indicate a risk of FDVrelated CNS effects. Pivotal enabling studies included (i) repeat dose toxicity studies in monkeys from 2 weeks to 9 months in duration achieving FDV systemic exposures 1 to 6 times the clinical efficacious exposure with no evidence of neurobehavioral signs or CNS histopathologic abnormalities and (ii) a quantitative whole body autoradiography study in mice suggesting minimal brain penetration or evidence of covalent binding in any part of the body (data not shown). In addition, there were no CNS effects in a rat neurobehavioral study or in the repeat dose mouse toxicity studies of up to 6 months in duration; however, the maximum feasible exposures in rodents were low relative to those needed for clinical efficacy. Although neurobehavioral signs were observed in a pregnant rabbit tolerability study, these were considered nonspecific at the time because they occurred at a dose where pronounced loss of body weight and marked reductions in food consumption indicated excessive maternal toxicity prohibitive for the conduct of the definitive rabbit embryo-fetal development (EFD) study. Indeed, the absence of neurobehavioral signs in the ensuing EFD study achieving similar systemic exposure using a split dosing schedule without excessive maternal toxicity appeared to confirm that conclusion. After FDV was placed on clinical hold due to seizure in patients on the phase IIb study, results from a single dose escalating tolerability study in minipigs became available. This study was conducted to identify a second nonrodent species capable of FDV systemic exposures greater than those required for clinical efficacy. One minipig that received escalating single doses of FDV (100, 500, and 1000 mg/kg) exhibited neurobehavioral signs within 2−4 h after each dose that resolved by 24 h. Three additional animals exhibited neurobehavioral signs 8−25 days after the last dose of 1000 mg/kg. The animals were euthanized, and the brain and spinal cord were processed for histopathologic examination, but no abnormalities were found. In this article, FDV metabolism and disposition in the CNS were investigated to determine whether differences among patients and preclinical species could explain the observed variability in neurobehavioral effects despite comparable plasma exposure. We report here data from a series of studies including (i) LC-MS analysis of CSF samples collected from patients with episodes of seizure up to 40 days postdose, (ii) characterization and concentration estimation of FDV and its metabolites by LC-MS in rabbits and monkeys (plasma, CSF, and brain) following repeat oral dosing, and (iii) brain tissue distribution of FDV and its metabolites in rabbits, monkeys, and minipigs using MALDI IMS.7−9
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MATERIALS AND METHODS
Materials. Fosdevirine (GSK2248761A, purity 99.7%) and [14C]fosdevirine (55.4 μCi/mol, radiochemical purity determined by HPLC with radiochemical detection was >99%) were synthesized by Isotope Chemistry, GlaxoSmithKline, Stevenage, UK.10,11 The synthetic scheme showing incorporation of the 14C-label is included in the Supporting Information. Biological reference standards for M6 (Odemethylated FDV) and M16 (O-demethylated cysteine conjugate of FDV) were isolated from human hepatocytes. M22 (cysteine conjugate of FDV) was isolated from minipig hepatocytes following incubations with [14C]FDV (27.7 μCi/mol). M42 (hydroxylamine of FDV) was isolated following the incubation of [14C]FDV (55.4 μCi/ 242
dx.doi.org/10.1021/tx3004196 | Chem. Res. Toxicol. 2013, 26, 241−251
Chemical Research in Toxicology
Article
A matrix solution consisting of 2,5-dihydroxybenzoic acid (20 mg/ mL) dissolved in water/methanol (1:1 v/v) was applied to the surface of the tissue using an ImagePrep automated spray device (Bruker Daltonics, Billerica, MA). A series of 200 spray cycles were completed to achieve a homogeneous coating of matrix on the tissue. Matrix assisted laser desorption/ionization (MALDI) IMS was performed using a Bruker Solarix 7T Fourier transform-ion cyclotron resonance mass spectrometer (Bruker Daltonics, Billerica, MA). Images were acquired at spatial resolutions ranging from 160 to 500 μm. In general, the MS data were collected in the mass range of m/z 350−650. In some cases, the mass spectrometer employed CASI (continuous accumulation of selected ions) where a narrow mass range (460−560) was analyzed to enhance sensitivity. Ion images were generated using FlexImaging v3.0 software from the raw data with a mass tolerance of ±0.005 Da.
Table 1. Estimated Concentrations of FDV-Related Material in Cerebral Spinal Fluid from Seizure Patients Following Repeat Oral Dosing
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RESULTS LC-MS Analysis of CSF from Seizure Patients. A summary of the FDV drug-related material (DRM) identified in CSF from seizure patients is presented in Table 1. The highest estimated concentration of FDV DRM was in the CSF sample collected 6 days after the last dose (Patient A, ∼39 pmol/mL), while the lowest levels were observed at 40 days after the last dose (Patient D, ∼3 pmol/mL). A total of 9 metabolites, in addition to FDV, were characterized in the CSF sample from Patient A. Three of these metabolites (M19, M23, and M38) had undergone oxidation and conjugation. Combined, M19, M23, and M38 represented
