RESUMEN
Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.
Asunto(s)
Progresión de la Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Leucemia Mieloide Aguda , Inhibidores de Prolil-Hidroxilasa , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Inhibidores de Prolil-Hidroxilasa/farmacología , Inhibidores de Prolil-Hidroxilasa/uso terapéutico , Animales , Ratones , Apoptosis/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Línea Celular Tumoral , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Estabilidad Proteica/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con PuentesRESUMEN
PURPOSE: We aim to present a refined thin-film model describing the drug particle dissolution considering radial diffusion in spherical boundary layer, and to demonstrate the ability of the model to describe the dissolution behavior of bulk drug powders. METHODS: The dissolution model introduced in this study was refined from a radial diffusion-based model previously published by our laboratory (So et al. in Pharm Res. 39:907-17, 2022). The refined model was created to simulate the dissolution of bulk powders, and to account for the evolution of particle size and diffusion layer thickness during dissolution. In vitro dissolution testing, using fractionated hydrochlorothiazide powders, was employed to assess the performance of the model. RESULTS: Overall, there was a good agreement between the experimental dissolution data and the predicted dissolution profiles using the proposed model across all size fractions of hydrochlorothiazide. The model over-predicted the dissolution rate when the particles became smaller. Notably, the classic Nernst-Brunner formalism led to an under-estimation of the dissolution rate. Additionally, calculation based on the equivalent particle size derived from the specific surface area substantially over-predicted the dissolution rate. CONCLUSION: The study demonstrated the potential of the radial diffusion-based model to describe dissolution of drug powders. In contrast, the classic Nernst-Brunner equation could under-estimate drug dissolution rate, largely due to the underlying assumption of translational diffusion. Moreover, the study indicated that not all surfaces on a drug particle contribute to dissolution. Therefore, relying on the experimentally-determined specific surface area for predicting drug dissolution is not advisable.
Asunto(s)
Liberación de Fármacos , Hidroclorotiazida , Tamaño de la Partícula , Polvos , Solubilidad , Polvos/química , Difusión , Hidroclorotiazida/química , Química Farmacéutica/métodos , Modelos Químicos , Simulación por ComputadorRESUMEN
ABSTRACT: Maintenance of quiescence and DNA replication dynamics are 2 paradoxical requirements for the distinct states of dormant and active hematopoietic stem cells (HSCs), which are required to preserve the stem cell reservoir and replenish the blood cell system in response to hematopoietic stress, respectively. Here, we show that key self-renewal factors, ß-catenin or Hoxa9, largely dispensable for HSC integrity, in fact, have dual functions in maintaining quiescence and enabling efficient DNA replication fork dynamics to preserve the functionality of hematopoietic stem and progenitor cells (HSPCs). Although ß-catenin or Hoxa9 single knockout (KO) exhibited mostly normal hematopoiesis, their coinactivation led to severe hematopoietic defects stemmed from aberrant cell cycle, DNA replication, and damage in HSPCs. Mechanistically, ß-catenin and Hoxa9 function in a compensatory manner to sustain key transcriptional programs that converge on the pivotal downstream target and epigenetic modifying enzyme, Prmt1, which protects the quiescent state and ensures an adequate supply of DNA replication and repair factors to maintain robust replication fork dynamics. Inactivation of Prmt1 phenocopied both cellular and molecular phenotypes of ß-catenin/Hoxa9 combined KO, which at the same time could also be partially rescued by Prmt1 expression. The discovery of the highly resilient ß-catenin/Hoxa9/Prmt1 axis in protecting both quiescence and DNA replication dynamics essential for HSCs at different key states provides not only novel mechanistic insights into their intricate regulation but also a potential tractable target for therapeutic intervention.
Asunto(s)
Células Madre Hematopoyéticas , beta Catenina , beta Catenina/metabolismo , Células Madre Hematopoyéticas/metabolismo , Ciclo Celular , División Celular , Replicación del ADNRESUMEN
Acute myeloid leukemia (AML) with TP53 mutation is one of the most lethal cancers and portends an extremely poor prognosis. Based on in silico analyses of druggable genes and differential gene expression in TP53-mutated AML, we identified pololike kinase 4 (PLK4) as a novel therapeutic target and examined its expression, regulation, pathogenetic mechanisms, and therapeutic potential in TP53-mutated AML. PLK4 expression was suppressed by activated p53 signaling in TP53 wild-type AML and was increased in TP53-mutated AML cell lines and primary samples. Short-term PLK4 inhibition induced DNA damage and apoptosis in TP53 wild-type AML. Prolonged PLK4 inhibition suppressed the growth of TP53-mutated AML and was associated with DNA damage, apoptosis, senescence, polyploidy, and defective cytokinesis. A hitherto undescribed PLK4/PRMT5/EZH2/H3K27me3 axis was demonstrated in both TP53 wild-type and mutated AML, resulting in histone modification through PLK4-induced PRMT5 phosphorylation. In TP53-mutated AML, combined effects of histone modification and polyploidy activated the cGAS-STING pathway, leading to secretion of cytokines and chemokines and activation of macrophages and T cells upon coculture with AML cells. In vivo, PLK4 inhibition also induced cytokine and chemokine expression in mouse recipients, and its combination with anti-CD47 antibody, which inhibited the "don't-eat-me" signal in macrophages, synergistically reduced leukemic burden and prolonged animal survival. The study shed important light on the pathogenetic role of PLK4 and might lead to novel therapeutic strategies in TP53-mutated AML.
Asunto(s)
Histonas , Leucemia Mieloide Aguda , Animales , Ratones , Histonas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Mutación , Metilación , Nucleotidiltransferasas/metabolismo , Leucemia Mieloide Aguda/patología , Inmunidad , PoliploidíaRESUMEN
The interactions between Acute Myeloid Leukaemia (AML) leukemic stem cells and the bone marrow (BM) microenvironment play a critical role during AML progression and resistance to drug treatments. Therefore, the identification of novel therapies requires drug-screening methods using in vitro co-culture models that closely recreate the cytoprotective BM setting. We have developed a new fluorescence-based in vitro co-culture system scalable to high throughput for measuring the concomitant effect of drugs on AML cells and the cytoprotective BM microenvironment. eGFP-expressing AML cells are co-cultured in direct contact with mCherry-expressing BM stromal cells for the accurate assessment of proliferation, viability, and signaling in both cell types. This model identified several efficacious compounds that overcome BM stroma-mediated drug resistance against daunorubicin, including the chromosome region maintenance 1 (CRM1/XPO1) inhibitor KPT-330. In silico analysis of genes co-expressed with CRM1, combined with in vitro experiments using our new methodology, also indicates that the combination of KPT-330 with the AURKA pharmacological inhibitor alisertib circumvents the cytoprotection of AML cells mediated by the BM stroma. This new experimental model and analysis provide a more precise screening method for developing improved therapeutics targeting AML cells within the cytoprotective BM microenvironment.
Asunto(s)
Medicina General , Neoplasias Cutáneas , Humanos , Victoria , Neoplasias Cutáneas/diagnóstico , BiopsiaRESUMEN
Acute myeloid leukaemia (AML) is a rapidly fatal blood cancer that is characterised by the accumulation of immature myeloid cells in the blood and bone marrow as a result of blocked differentiation. Methods which identify master transcriptional regulators of AML subtype-specific leukaemia cell states and their combinations could be critical for discovering novel differentiation-inducing therapies. In this proof-of-concept study, we demonstrate a novel utility of the Mogrify® algorithm in identifying combinations of transcription factors (TFs) and drugs, which recapitulate granulocytic differentiation of the NB4 acute promyelocytic leukaemia (APL) cell line, using two different approaches. In the first approach, Connectivity Map (CMAP) analysis of these TFs and their target networks outperformed standard approaches, retrieving ATRA as the top hit. We identify dimaprit and mebendazole as a drug combination which induces myeloid differentiation. In the second approach, we show that genetic manipulation of specific Mogrify®-identified TFs (MYC and IRF1) leads to co-operative induction of APL differentiation, as does pharmacological targeting of these TFs using currently available compounds. We also show that loss of IRF1 blunts ATRA-mediated differentiation, and that MYC represses IRF1 expression through recruitment of PML-RARα, the driver fusion oncoprotein in APL, to the IRF1 promoter. Finally, we demonstrate that these drug combinations can also induce differentiation of primary patient-derived APL cells, and highlight the potential of targeting MYC and IRF1 in high-risk APL. Thus, these results suggest that Mogrify® could be used for drug discovery or repositioning in leukaemia differentiation therapy for other subtypes of leukaemia or cancers.
Asunto(s)
Leucemia Mieloide Aguda , Leucemia Promielocítica Aguda , Humanos , Tretinoina/farmacología , Tretinoina/uso terapéutico , Farmacología en Red , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Diferenciación Celular/genética , Factores de Transcripción/genéticaRESUMEN
Despite the expanding portfolio of targeted therapies for adults with acute myeloid leukemia (AML), direct implementation in children is challenging due to inherent differences in underlying genetics. Here we established the pharmacologic profile of pediatric AML by screening myeloblast sensitivity to approved and investigational agents, revealing candidates of immediate clinical relevance. Drug responses ex vivo correlated with patient characteristics, exhibited age-specific alterations, and concorded with activities in xenograft models. Integration with genomic data uncovered new gene-drug associations, suggesting actionable therapeutic vulnerabilities. Transcriptome profiling further identified gene-expression signatures associated with on- and off-target drug responses. We also demonstrated the feasibility of drug screening-guided treatment for children with high-risk AML, with two evaluable cases achieving remission. Collectively, this study offers a high-dimensional gene-drug clinical data set that could be leveraged to research the unique biology of pediatric AML and sets the stage for realizing functional precision medicine for the clinical management of the disease. SIGNIFICANCE: We conducted integrated drug and genomic profiling of patient biopsies to build the functional genomic landscape of pediatric AML. Age-specific differences in drug response and new gene-drug interactions were identified. The feasibility of functional precision medicine-guided management of children with high-risk AML was successfully demonstrated in two evaluable clinical cases. This article is highlighted in the In This Issue feature, p. 476.
Asunto(s)
Leucemia Mieloide Aguda , Medicina de Precisión , Niño , Adulto , Humanos , Medicina de Precisión/métodos , Farmacogenética , Leucemia Mieloide Aguda/tratamiento farmacológico , Perfilación de la Expresión Génica/métodos , TranscriptomaRESUMEN
Thalassemia is one of the most common genetic diseases and a major health threat worldwide. Accurate, efficient, and scalable analysis of next-generation sequencing (NGS) data is much needed for its molecular diagnosis and carrier screening. We developed NGS4THAL, a bioinformatics analysis pipeline analyzing NGS data to detect pathogenic variants for thalassemia and other hemoglobinopathies. NGS4THAL realigns ambiguously mapped NGS reads derived from the homologous Hb gene clusters for accurate detection of point mutations and small insertions/deletions. It uses a combination of complementary structural variant (SV) detection tools and an in-house database of control data containing specific SVs to achieve accurate detection of the complex SV types. Detected variants are matched with those in HbVar (A Database of Human Hemoglobin Variants and Thalassemia Mutations), allowing recognition of known pathogenic variants, including disease modifiers. Tested on simulation data, NGS4THAL achieved high sensitivity and specificity. For targeted NGS sequencing data from samples with laboratory-confirmed pathogenic Hb variants, it achieved 100% detection accuracy. Application of NGS4THAL on whole genome sequencing data from unrelated studies revealed thalassemia mutation carrier rates for Hong Kong Chinese and Northern Vietnamese that were consistent with previous reports. NGS4THAL is a highly accurate and efficient molecular diagnosis tool for thalassemia and other hemoglobinopathies based on tailored analysis of NGS data and may be scaled for population carrier screening.
Asunto(s)
Hemoglobinopatías , Talasemia , Hemoglobinopatías/diagnóstico , Hemoglobinopatías/epidemiología , Hemoglobinopatías/genética , Hemoglobinas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Talasemia/diagnóstico , Talasemia/genéticaRESUMEN
Pharmaceutical products intended for parenteral use must be free from pyrogenic (fever-inducing) contamination. Pyrogens comprise endotoxins from Gram-negative bacteria and non-endotoxin pyrogens from Gram-positive bacteria, viruses, and fungi. The longstanding compendial test for pyrogens is the rabbit pyrogen test, but in 2010 the monocyte acti-vation test (MAT) for pyrogenic and pro-inflammatory contaminants was introduced into the European Pharmacopoeia (Ph. Eur.) as a non-animal replacement for the rabbit pyrogen test. The present study describes the first product-specific Good Manufacturing Practice validation of Ph. Eur. MAT, Quantitative Test, Method A for the testing of three therapeutic monoclonal antibodies. The study used the MAT version with cryo-preserved peripheral blood mononuclear cells and interleukin-6 as the readout. Much of the data presented here for one of the antibodies was included in a successful product license application to the European Medicines Agency.
Asunto(s)
Monocitos , Pirógenos , Animales , Conejos , Anticuerpos Monoclonales/farmacología , Leucocitos Mononucleares , Alternativas a las Pruebas en Animales , EndotoxinasRESUMEN
Assessment and understanding of changes in particle size of active pharmaceutical ingredients (API) and excipients as a function of solid dosage form processing is an important but under-investigated area that can impact drug product quality. In this study, X-ray microscopy (XRM) was investigated as a method for determining the in situ particle size distribution of API agglomerates and an excipient at different processing stages in tablet manufacturing. An artificial intelligence (AI)-facilitated XRM image analysis tool was applied for quantitative analysis of thousands of individual particles, both of the API and the major filler component of the formulation, microcrystalline cellulose (MCC). Domain size distributions for API and MCC were generated along with the calculation of the porosity of each respective component. The API domain size distributions correlated with laser diffraction measurements and sieve analysis of the API, formulation blend, and granulation. The XRM analysis demonstrated that attrition of the API agglomerates occurred secondary to the granulation stage. These results were corroborated by particle size distribution and sieve potency data which showed generation of an API fines fraction. Additionally, changes in the XRM-calculated size distribution of MCC particles in subsequent processing steps were rationalized based on the known plastic deformation mechanism of MCC. The XRM data indicated that size distribution of the primary MCC particles, which make up the larger functional MCC agglomerates, is conserved across the stages of processing. The results indicate that XRM can be successfully applied as a direct, non-invasive method to track API and excipient particle properties and microstructure for in-process control samples and in the final solid dosage form. The XRM and AI image analysis methodology provides a data-rich way to interrogate the impact of processing stresses on API and excipients for enhanced process understanding and utilization for Quality by Design (QbD).
Asunto(s)
Excipientes , Microscopía , Inteligencia Artificial , Excipientes/química , Tamaño de la Partícula , Comprimidos , Rayos XRESUMEN
PURPOSE: The purpose of the study is to present a mathematical model capable of describing drug particle dissolution in 3-dimensional (3D) space, and to provide experimental model verification. Through this study, we also aim to elaborate limitations of the classic, 1D-based Nernst-Brunner formalism in dissolution modeling. METHODS: The 3D dissolution model was derived by treating the dissolution of a spherical particle as a diffusion-driven process, and by solving Fick's 2nd law of diffusion in spherical coordinates using numerical methods. The resulting model was experimentally verified through analyzing the dissolution behavior of single succinic acid particles in un-stirred water droplet under polarized light microscopy, in combination with image segmentation techniques. RESULTS: A set of working equations was developed to describe drug particle dissolution in 3D space. The predicted dissolution time and profile are in good agreement with the experimental results. The model clearly shows that the concentration gradient within the diffusion layer, in realistic 3D condition, must not be a constant value as implicated in the Nernst-Brunner formalism. The actual concentration profile is a hyperbola, and the concentration gradient at the surface of the particle can be significantly higher than the classic 1D-based dissolution model. CONCLUSION: The study demonstrates that the classic, 1D-based dissolution models may lead to significant under-estimation of drug dissolution rates. In contrast, modeling dissolution in 3D space yields more reliable results. This study merits further development of comprehensive 3D drug dissolution models, by considering polydispersed particle ensemble and imposing the changes of diffusion layer thickness during dissolution.
Asunto(s)
Modelos Teóricos , Agua , Difusión , Liberación de Fármacos , SolubilidadRESUMEN
HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of ß-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced ß-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.
Asunto(s)
Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Leucemia Mieloide Aguda/metabolismo , Estructuras R-Loop , ARN Largo no Codificante/metabolismo , beta Catenina/metabolismo , Animales , Factor de Unión a CCCTC/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación Leucémica de la Expresión Génica , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones Transgénicos , ARN Largo no Codificante/genética , Relación Estructura-Actividad , Transcripción Genética , Activación Transcripcional , beta Catenina/genética , CohesinasRESUMEN
Posttranslational modification of histones serve a crucial role in the control of gene transcription. Trimethylation of lysine 4 on histone 3 is associated with transcription activation. There are currently six known methylases and six known demethylases that can control the methylation status of this site. Lysine demethylase 5B (KDM5B) is one such demethylase, which can repress gene expression. In particular KDM5B has been found to be overexpressed in a number of cancer types, and smallmolecular weight inhibitors of its demethylase activity have been identified. Previous characterisation of Kdm5b knockout mice has revealed that this genotype leads to either embryonic or neonatal lethality. However, the ΔAT rich interaction domain (ΔARID)KDM5B strain of mice, which have the ARID domain and five amino acids within the Jumonji (Jmj)N domain spliced out from KDM5B, remain viable and fertile. In the present study, ΔARIDKDM5B was found to have no demethylase activity as determined by in vitro demethylase assays and by immunofluorescence in transfected Cos1 cells. Furthermore, molecular dynamic simulations revealed conformational changes within the ΔARIDKDM5B structure compared with that in WTKDM5B, particularly in the JmjC domain, which is responsible for the catalytic activity of WTKDM5B. This supports the experimental data that shows the loss of demethylase activity. Since Kdm5b knockout mice show varying degrees of lethality, these data suggest that KDM5B serves a crucial function in development in a manner that is independent of its demethylase activity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Dominios Proteicos/genética , Animales , Desmetilación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Pruebas de Enzimas , Femenino , Fertilidad/genética , Regulación del Desarrollo de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/ultraestructura , Masculino , Ratones , Ratones Transgénicos , Simulación de Dinámica Molecular , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
The purpose of the study is to present the finite difference method (FDM) and demonstrate its utility in modeling mass transport processes that are pharmaceutically relevant. In particular, diffusion processes are ideally suited for FDM because the governing equation, Fick's second law of diffusion, can be readily solved using FDM over a finite space and time. The method entails the mesh creation, space and time discretization, and solving Fick's second law at each node using finite difference-based numerical schemes. We applied FDM to study tablet disintegration, in which the tablet water uptake was simulated with an effective water diffusion coefficient; the tablet disintegration was controlled by a designated critical water content parameter, beyond which the node is treated as being disintegrated from the tablet. The resulting simulation agreed with the experimental tablet disintegration behaviors, under both disintegration-controlled and water uptake-controlled conditions. This study highlighted the unique advantage of FDM, capable of providing spatial-temporal information on water uptake and evolution of tablet size and shape during tablet disintegration, which was otherwise not available using other methods. The FDM method enabled more in-depth tablet disintegration studies. The model also has the potential to be calibrated and incorporated in tablet formulation DoE studies.
Asunto(s)
Agua , Difusión , Solubilidad , ComprimidosRESUMEN
Transcriptional deregulation is a key driver of acute myeloid leukemia (AML), a heterogeneous blood cancer with poor survival rates. Polycomb group (PcG) and Trithorax group (TrxG) genes, originally identified in Drosophila melanogaster several decades ago as master regulators of cellular identity and epigenetic memory, not only are important in mammalian development but also play a key role in AML disease biology. In addition to their classical canonical antagonistic transcriptional functions, noncanonical synergistic and nontranscriptional functions of PcG and TrxG are emerging. Here, we review the biochemical properties of major mammalian PcG and TrxG complexes and their roles in AML disease biology, including disease maintenance as well as drug resistance. We summarize current efforts on targeting PcG and TrxG for treatment of AML and propose rational synthetic lethality and drug-induced antagonistic pleiotropy options involving PcG and TrxG as potential new therapeutic avenues for treatment of AML.
Asunto(s)
Drosophila melanogaster , Leucemia Mieloide Aguda , Animales , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas del Grupo Polycomb/genéticaRESUMEN
The purpose of the study is to build a "virtual roller compactor" as a predictive tool to assess the roll force (RF)-maximum pressure (Pmax) and RF-ribbon density relationship for pharmaceutical roller compaction. We provided a theoretical basis to demonstrate that, there exists a critical nip angle for a pharmaceutical powder, beyond which the RF-Pmax relationship is insensitive to wall friction angle or effective angle of internal friction. We showed that for most pharmaceutical roller compaction, the critical nip angle is lower than 17 degree, and can be exceeded via wall friction elevation, using rolls with non-smooth surface. Under this condition, the original Johanson model can be substantially simplified to a single equation requiring only one material property (compressibility). By performing manufacturing-scale roller compaction using materials with diverse compressibility, we showed that the simplified, friction angle-free model performed similar to the original Johanson model. It can predict the RF-Pmax and RF-ribbon density relationship well after applying a correction factor. The predictive tool, in the form of a user-friendly graphical user interface, was created based on the simplified model. The tool was adopted for in-house, bench-scale formulation development and scale-up because of its ease-of-use, good predicting capability, and very low material demand.
Asunto(s)
Tecnología Farmacéutica , Composición de Medicamentos , Fricción , Tamaño de la Partícula , Polvos , ComprimidosRESUMEN
In tablet manufacturing, mixing operations in tumble blending (TB) and in the feed frame (FF) of the rotary press can both increase lubricity, negatively influencing the tablet mechanical strength. While the TB-driven lubrication was systematically studied, no reliable bench-scale methods exist for the effect of FF lubrication. Because TB and FF mixing are usually two successive operations in tablet manufacturing, we developed a phenomenological model to incorporate the impact of TB-driven lubrication and the FF lubrication on the tablet tensile strength (TS). We noted that exponential decay functions can describe the evolution of the tablet TS as the function of the extent of TB, as well as the residence time in FF. Hence, the overall lubrication sensitivity can be modeled by incorporating two distinct exponential decay functions. The model can be calibrated through bench-scale experiments. Using an investigational powder blend, we showed that this approach accurately predicted the tablet TS in a scale-up tablet compression study, thereby verifying its utility. This model can serve as a scale-up diagnostic and risk-assessment tool, with the ability to adjust the overall effect of lubrication by changing the TB scale and the FF residence time commensurate with the large-scale operations.
Asunto(s)
Lubrificación , Composición de Medicamentos , Polvos , Comprimidos , Resistencia a la TracciónRESUMEN
Chemoresistance remains the major challenge for successful treatment of acute myeloid leukemia (AML). Although recent mouse studies suggest that treatment response of genetically and immunophenotypically indistinguishable AML can be influenced by their different cells of origin, corresponding evidence in human disease is still largely lacking. By combining prospective disease modeling using highly purified human hematopoietic stem or progenitor cells with retrospective deconvolution study of leukemia stem cells (LSCs) from primary patient samples, we identified human hematopoietic stem cells (HSCs) and common myeloid progenitors (CMPs) as two distinctive origins of human AML driven by Mixed Lineage Leukemia (MLL) gene fusions (MLL-AML). Despite LSCs from either MLL-rearranged HSCs or MLL-rearranged CMPs having a mature CD34-/lo/CD38+ immunophenotype in both a humanized mouse model and primary patient samples, the resulting AML cells exhibited contrasting responses to chemotherapy. HSC-derived MLL-AML was highly resistant to chemotherapy and expressed elevated amounts of the multispecific anion transporter ABCC3. Inhibition of ABCC3 by shRNA-mediated knockdown or with small-molecule inhibitor fidaxomicin, currently used for diarrhea associated with Clostridium difficile infection, effectively resensitized HSC-derived MLL-AML toward standard chemotherapeutic drugs. This study not only functionally established two distinctive origins of human LSCs for MLL-AML and their role in mediating chemoresistance but also identified a potential therapeutic avenue for stem cell-associated treatment resistance by repurposing a well-tolerated antidiarrhea drug already used in the clinic.