ABSTRACT
Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualification plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharmaceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.
Subject(s)
Animal Testing Alternatives , Lab-On-A-Chip Devices , Animals , Drug Evaluation, Preclinical , Drug Industry , Pharmaceutical Preparations/metabolism , United States , United States Food and Drug AdministrationABSTRACT
The liver is critical to consider during drug development because of its central role in the handling of xenobiotics, a process which often leads to localized and/or downstream tissue injury. Our ability to predict human clinical safety outcomes with animal testing is limited due to species differences in drug metabolism and disposition, while traditional human in vitro liver models often lack the necessary in vivo physiological fidelity. To address this, increasing numbers of liver microphysiological systems (MPS) are being developed, however the inconsistency in their optimization and characterization often leads to models that do not possess critical levels of baseline performance that is required for many pharmaceutical industry applications. Herein we provide a guidance on best approaches to benchmark liver MPS based on 3 stages of characterization that includes key performance metrics and a 20 compound safety test set. Additionally, we give an overview of frequently used liver injury safety assays, describe the ideal MPS model, and provide a perspective on currently best suited MPS contexts of use. This pharmaceutical industry guidance has been written to help MPS developers and end users identify what could be the most valuable models for safety risk assessment.
Subject(s)
Liver/metabolism , Pharmaceutical Preparations/metabolism , Animals , Drug Evaluation, Preclinical , Drug Industry , Humans , Lab-On-A-Chip Devices , Liver/chemistry , Pharmaceutical Preparations/chemistry , Risk AssessmentABSTRACT
Although glycolysis is substantially elevated in many tumors, therapeutic targeting of glycolysis in cancer patients has not yet been successful, potentially reflecting the metabolic plasticity of tumor cells. In various cancer cells exposed to a continuous glycolytic block, we identified a recurrent reprogramming mechanism involving sustained mTORC1 signaling that underlies escape from glycolytic addiction. Active mTORC1 directs increased glucose flux via the pentose phosphate pathway back into glycolysis, thereby circumventing a glycolysis block and ensuring adequate ATP and biomass production. Combined inhibition of glycolysis and mTORC1 signaling disrupted metabolic reprogramming in tumor cells and inhibited their growth in vitro and in vivo. These findings reveal novel combinatorial therapeutic strategies to realize the potential benefit from targeting the Warburg effect.
Subject(s)
Glycolysis , Molecular Targeted Therapy , Multiprotein Complexes/physiology , Neoplasm Proteins/physiology , Neoplasms/metabolism , TOR Serine-Threonine Kinases/physiology , Adenosine Triphosphate/biosynthesis , Animals , Carcinoma/pathology , Cell Line, Tumor , Citric Acid Cycle , Combined Modality Therapy , Cytokines/antagonists & inhibitors , Cytokines/genetics , Deoxyglucose/pharmacology , Deoxyglucose/therapeutic use , Drug Resistance, Neoplasm , Drug Synergism , Energy Metabolism/drug effects , Everolimus/pharmacology , Everolimus/therapeutic use , Female , Glucose-6-Phosphate Isomerase/antagonists & inhibitors , Glucose-6-Phosphate Isomerase/genetics , Glutaminase/antagonists & inhibitors , Glutaminase/physiology , Glutamine/metabolism , Glycolysis/drug effects , Hep G2 Cells , Humans , Mechanistic Target of Rapamycin Complex 1 , Metabolomics , Mice , Mice, Nude , Multiprotein Complexes/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Pentose Phosphate Pathway/drug effects , Pentose Phosphate Pathway/physiology , RNA Interference , RNA, Small Interfering/therapeutic use , Ribosomal Protein S6 Kinases, 70-kDa/antagonists & inhibitors , Ribosomal Protein S6 Kinases, 70-kDa/physiology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Tumor Stem Cell Assay , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: We recently showed improved between-subject variability in our [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) experiments using a Michaelis-Menten transport model to calculate the metabolic tumor glucose uptake rate extrapolated to the hypothetical condition of glucose saturation: MRglucmax=Ki*(KM+[glc]), where Ki is the image-derived FDG uptake rate constant, KM is the half-saturation Michaelis constant, and [glc] is the blood glucose concentration. Compared to measurements of Ki alone, or calculations of the scan-time metabolic glucose uptake rate (MRgluc = Ki * [glc]) or the glucose-normalized uptake rate (MRgluc = Ki*[glc]/(100 mg/dL), we suggested that MRglucmax could offer increased statistical power in treatment studies; here, we confirm this in theory and practice. METHODS: We compared Ki, MRgluc (both with and without glucose normalization), and MRglucmax as FDG-PET measures of treatment-induced changes in tumor glucose uptake independent of any systemic changes in blood glucose caused either by natural variation or by side effects of drug action. Data from three xenograft models with independent evidence of altered tumor cell glucose uptake were studied and generalized with statistical simulations and mathematical derivations. To obtain representative simulation parameters, we studied the distributions of Ki from FDG-PET scans and blood [glucose] values in 66 cohorts of mice (665 individual mice). Treatment effects were simulated by varying MRglucmax and back-calculating the mean Ki under the Michaelis-Menten model with KM = 130 mg/dL. This was repeated to represent cases of low, average, and high variability in Ki (at a given glucose level) observed among the 66 PET cohorts. RESULTS: There was excellent agreement between derivations, simulations, and experiments. Even modestly different (20%) blood glucose levels caused Ki and especially MRgluc to become unreliable through false positive results while MRglucmax remained unbiased. The greatest benefit occurred when Ki measurements (at a given glucose level) had low variability. Even when the power benefit was negligible, the use of MRglucmax carried no statistical penalty. Congruent with theory and simulations, MRglucmax showed in our experiments an average 21% statistical power improvement with respect to MRgluc and 10% with respect to Ki (approximately 20% savings in sample size). The results were robust in the face of imprecise blood glucose measurements and KM values. CONCLUSIONS: When evaluating the direct effects of treatment on tumor tissue with FDG-PET, employing a Michaelis-Menten glucose correction factor gives the most statistically powerful results. The well-known alternative 'correction', multiplying Ki by blood glucose (or normalized blood glucose), appears to be counter-productive in this setting and should be avoided.
ABSTRACT
Glucocorticoids are standard of care for many inflammatory conditions, but chronic use is associated with a broad array of side effects. This has led to a search for dissociative glucocorticoids--drugs able to retain or improve efficacy associated with transrepression [nuclear factor-κB (NF-κB) inhibition] but with the loss of side effects associated with transactivation (receptor-mediated transcriptional activation through glucocorticoid response element gene promoter elements). We investigated a glucocorticoid derivative with a Δ-9,11 modification as a dissociative steroid. The Δ-9,11 analog showed potent inhibition of tumor necrosis factor-α-induced NF-κB signaling in cell reporter assays, and this transrepression activity was blocked by 17ß-hydroxy-11ß-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU-486), showing the requirement for the glucocorticoid receptor (GR). The Δ-9,11 analog induced the nuclear translocation of GR but showed the loss of transactivation as assayed by GR-luciferase constructs as well as mRNA profiles of treated cells. The Δ-9,11 analog was tested for efficacy and side effects in two mouse models of muscular dystrophy: mdx (dystrophin deficiency), and SJL (dysferlin deficiency). Daily oral delivery of the Δ-9,11 analog showed a reduction of muscle inflammation and improvements in multiple muscle function assays yet no reductions in body weight or spleen size, suggesting the loss of key side effects. Our data demonstrate that a Δ-9,11 analog dissociates the GR-mediated transcriptional activities from anti-inflammatory activities. Accordingly, Δ-9,11 analogs may hold promise as a source of safer therapeutic agents for chronic inflammatory disorders.
Subject(s)
Dronabinol/analogs & derivatives , Glucocorticoids/adverse effects , Glucocorticoids/pharmacology , NF-kappa B/antagonists & inhibitors , Response Elements/drug effects , Animals , Dose-Response Relationship, Drug , Dronabinol/chemistry , Dronabinol/pharmacology , Female , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Mice, Knockout , NF-kappa B/metabolism , Response Elements/physiology , Spleen/drug effects , Spleen/metabolism , Treatment OutcomeABSTRACT
BACKGROUND: The BRAF inhibitor, vemurafenib, has recently been approved for the treatment of metastatic melanoma in patients harboring BRAFV600 mutations. Currently, dual BRAF and MEK inhibition are ongoing in clinical trials with the goal of overcoming the acquired resistance that has unfortunately developed in some vemurafenib patients. FDG-PET measures of metabolic activity are increasingly employed as a pharmacodynamic biomarker for guiding single-agent or combination therapies by gauging initial drug response and monitoring disease progression. However, since tumors are inherently heterogeneous, investigating the effects of BRAF and MEK inhibition on FDG uptake in a panel of different melanomas could help interpret imaging outcomes. METHODS: 18 F-FDG uptake was measured in vitro in cells with wild-type and mutant (V600) BRAF, and in melanoma cells with an acquired resistance to vemurafenib. We treated the cells with vemurafenib alone or in combination with MEK inhibitor GDC-0973. PET imaging was used in mice to measure FDG uptake in A375 melanoma xenografts and in A375 R1, a vemurafenib-resistant derivative. Histological and biochemical studies of glucose transporters, the MAPK and glycolytic pathways were also undertaken. RESULTS: We demonstrate that vemurafenib is equally effective at reducing FDG uptake in cell lines harboring either heterozygous or homozygous BRAFV600 but ineffective in cells with acquired resistance or having WT BRAF status. However, combination with GDC-0973 results in a highly significant increase of efficacy and inhibition of FDG uptake across all twenty lines. Drug-induced changes in FDG uptake were associated with altered levels of membrane GLUT-1, and cell lines harboring RAS mutations displayed enhanced FDG uptake upon exposure to vemurafenib. Interestingly, we found that vemurafenib treatment in mice bearing drug-resistant A375 xenografts also induced increased FDG tumor uptake, accompanied by increases in Hif-1α, Sp1 and Ksr protein levels. Vemurafenib and GDC-0973 combination efficacy was associated with decreased levels of hexokinase II, c-RAF, Ksr and p-MEK protein. CONCLUSIONS: We have demonstrated that 18 F-FDG-PET imaging reflects vemurafenib and GDC-0973 action across a wide range of metastatic melanomas. A delayed post-treatment increase in tumor FDG uptake should be considered carefully as it may well be an indication of acquired drug resistance. TRIAL REGISTRATION: ClinicalTrials.gov NCT01271803.
ABSTRACT
The identification of the Duchenne muscular dystrophy gene and protein in the late 1980s led to high hopes of rapid translation to molecular therapeutics. These hopes were fueled by early reports of delivering new functional genes to dystrophic muscle in mouse models using gene therapy and stem cell transplantation. However, significant barriers have thwarted translation of these approaches to true therapies, including insufficient therapeutic material (eg, cells and viral vectors), challenges in systemic delivery, and immunological hurdles. An alternative approach is to repair the patient's own gene. Two innovative small-molecule approaches have emerged as front-line molecular therapeutics: exon skipping and stop codon read through. Both approaches are in human clinical trials and aim to coax dystrophin protein production from otherwise inactive mutant genes. In the clinically severe dog model of Duchenne muscular dystrophy, the exon-skipping approach recently improved multiple functional outcomes. We discuss the status of these two methods aimed at inducing de novo dystrophin production from mutant genes and review implications for other disorders.
Subject(s)
Codon, Terminator/genetics , Dystrophin/metabolism , Exons/genetics , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/therapy , Mutant Proteins/metabolism , Animals , Dogs , Dystrophin/genetics , Humans , Mice , Muscle, Skeletal/cytology , Muscular Dystrophy, Duchenne/genetics , Mutant Proteins/geneticsABSTRACT
PURPOSE: To develop a reliable live-animal imaging method for monitoring muscle pathology in mouse models of myopathy. PROCEDURES: A caged near-infrared Cathepsin B (CTSB) substrate, ProSense 680, is evaluated in the dystrophin deficient mdx mice, a genetic homologue of Duchenne muscular dystrophy via optical imaging. RESULTS: We show high levels of infrared signal in dystrophic muscle relative to healthy muscle at 24 h post-injection. Imaging for CTSB presence revealed localization to inflammatory infiltrates and regenerating muscle fibers. A time series myotoxin-induced muscle injury experiment showed that CTSB activity and its mRNA levels peaked at the interface between inflammation and myoblast fusion stage of recovery. Prednisone treatment in mdx mice resulted in decreased CTSB activity and increased grip strength in forelimbs and hindlimbs. CONCLUSIONS: Optical imaging of CTSB activity is an ideal method to sensitively monitor inflammation, regeneration, and response to therapy in myopathic skeletal muscle.
Subject(s)
Cathepsin B/metabolism , Diagnostic Imaging/methods , Infrared Rays , Muscles/pathology , Optical Phenomena , Animals , Cathepsin B/genetics , Hindlimb/drug effects , Hindlimb/pathology , Humans , Injections, Intraperitoneal , Mice , Mice, Inbred mdx , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscles/drug effects , Muscles/enzymology , Muscular Dystrophy, Animal/pathology , Muscular Dystrophy, Duchenne/pathology , Myoblasts/drug effects , Myoblasts/metabolism , Myoblasts/pathology , Prednisone/pharmacology , Protein Transport/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regeneration/drug effects , Substrate Specificity/drug effects , Toxins, Biological/toxicity , Transcription, Genetic/drug effectsABSTRACT
Specific therapies are not available for inflammatory muscle diseases. We and others have shown that the pro-inflammatory NF-kappaB pathway is highly activated in these conditions. Since NF-kappaB is an important therapeutic target, we decided to utilize an in vitro screening assay to identify potential inhibitors that block TNF-alpha induced NF-kappaB activation in a C2C12 muscle line stably expressing an NF-kappaB luciferase reporter gene. Upon evaluation of multiple anti-inflammatory agents in undifferentiated myoblasts as well as differentiated myotubes , we found different levels of inhibition depending on the state of differentiation. Interestingly, we found that some drugs that are known to inhibit NF-kappaB in immune cells were not effective in muscle cells. Drug toxicity was assessed for using an MTT cell viability assay, and the validity of the luciferase assay was verified by immunostaining for NF-kappaB nuclear translocation in myoblasts. In conclusion, we have determined the optimal assay conditions for detecting potentially valuable NF-kappaB inhibitors for the first time in a muscle cell line that may have significant therapeutic potential for inflammatory muscle diseases.