RESUMO
A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here, we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily ("8HUM") can circumvent these problems. The pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani, and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery.
Assuntos
Doenças Transmissíveis , Descoberta de Drogas , Camundongos , Animais , Interações Medicamentosas , Modelos Animais de Doenças , Sistema Enzimático do Citocromo P-450/metabolismo , AceleraçãoRESUMO
Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.
Assuntos
Antiprotozoários/administração & dosagem , Leishmania donovani/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/diagnóstico por imagem , Inibidores de Proteassoma/administração & dosagem , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/química , Sítios de Ligação , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Leishmania donovani/química , Leishmania donovani/enzimologia , Leishmania infantum/química , Leishmania infantum/enzimologia , Leishmaniose Visceral/parasitologia , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismoRESUMO
In this study, rat models of wound closure by first and second intention were developed to evaluate the influence that two immunosuppressants for treating multiple sclerosis (fingolimod, azathioprine) have on wound healing. Sixty-three Sprague-Dawley rats were daily treated with fingolimod (0.6 mg/kg), azathioprine (2.5 mg/kg), or placebo (saline). Following 6 weeks of treatment, a linear incision (1.5 cm) or a circular excisional defect (diameter 1.5 cm) was made on the dorsal skin. The treatments were uninterrupted and after 7 days (incisional) or 21 days (incisional, excisional), animals were euthanized (n = 7 per group and time-point). Morphometric (wound closure), histological (stainings), and immunofluorescent studies (macrophages) were performed to evaluate the healing process. For both the incisional and excisional defects, animals treated with fingolimod exhibited a healing process equivalent to that of placebo in terms of collagenization, wound closure, and macrophage response. By comparison, groups treated with azathioprine displayed a delay in healing times which was especially evident in the excisional defect, where inflammatory reaction and collagen deposition in the repair tissue remained active by day 21. These results show that immunosuppressants with a selective mechanism of action (fingolimod) can have less impact on wound healing than their classical nonselective counterparts (azathioprine).
Assuntos
Azatioprina/farmacologia , Cloridrato de Fingolimode/farmacologia , Imunossupressores/farmacologia , Inflamação/tratamento farmacológico , Técnicas de Fechamento de Ferimentos , Cicatrização/efeitos dos fármacos , Animais , Inflamação/patologia , Masculino , Modelos Animais , Ratos , Ratos Sprague-DawleyRESUMO
A scaffold hopping strategy, including intellectual property availability assessment, was successfully applied for the discovery of novel PI3K inhibitors. Compounds were designed based on the chemical structure of the lead compound ETP-46321, a potent PI3K inhibitor, previously reported by our group. The new generated compounds showed good in vitro potency and selectivity, proved to inhibit potently the phosphorylation of AKTSer473 in cells and demonstrated to be orally bioavailable, thus becoming potential back-up candidates for ETP-46321.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/metabolismo , Administração Oral , Animais , Química Farmacêutica , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Imidazóis/química , Imidazóis/metabolismo , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazinas/química , Pirazinas/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismoRESUMO
The involvement of the phosphoinositide 3-kinases (PI3Ks) in several diseases, especially in the oncology area, has singled it as one of the most explored therapeutic targets in the last two decades. Many different inhibitor classes have been developed by the industry and academia with a diverse selectivity profile within the PI3K family. In the present manuscript we report a further exploration of our lead PI3K inhibitor ETP-46321 (Martínez González et al., 2012)1 by the application of a conformational restriction strategy. For that purpose we have successfully synthesized novel tricyclic imidazo[1,2-a]pyrazine derivatives as PI3K inhibitors. This new class of compounds had enable the exploration of the solvent-accessible region within PI3K and resulted in the identification of molecule 8q with the best selectivity PI3Kα/δ isoform profile in vitro, and promising in vivo PK data.
Assuntos
Imidazóis/química , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Pirazinas/química , Animais , Meia-Vida , Humanos , Imidazóis/síntese química , Imidazóis/farmacocinética , Concentração Inibidora 50 , Camundongos , Microssomos Hepáticos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Pirazinas/síntese química , Pirazinas/farmacocinética , Relação Estrutura-AtividadeRESUMO
Inhibitors of PI3K signaling are of great therapeutic interest in oncology. The phosphoinositide-3-kinase signaling pathway is activated in a variety of solid and non-solid tumors. We have identified an imidazopyrazine derivative, ETP-46321, as a potent inhibitor of PI3Kα [Formula: see text]. The compound was 6 times less potent towards PI3Kδ and more than 200 and 60 times less potent at inhibiting PI3Kß and PI3Kγ and did not significantly inhibit the related phosphoinositide-3-kinase-related protein kinase family kinases mTOR or DNA PK (IC(50)'s > 5 µM), or an additional 287 protein kinases that were screened. ETP-46321 inhibited PI3K signaling in treated tumor cell lines, induced cell cycle arrest and inhibited VEGF-dependent sprouting of HUVEC cells. The compound was anti-proliferative and synergized with both cytotoxic and targeted therapeutics. The compound induced a reduction in the phosphorylation of Akt in U87 MG xenografts after a single treatment. The growth of colon and lung cancinoma HT-29 and A549 xenografts was delayed by once a day treatment with ETP-46321. The compound synergized with Doxotaxel in a model of ovarian cancer.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Imidazóis/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Pirazinas/uso terapêutico , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imidazóis/sangue , Imidazóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Fosfatidilinositol 3-Quinases/metabolismo , Pirazinas/sangue , Pirazinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PIM kinases have become targets of interest due to their association with biochemical mechanisms affecting survival, proliferation and cytokine production. 1,2,3-Triazolo[4,5-b]pyridines were identified as PIM inhibitors applying a scaffold hopping approach. Initial exploration around this scaffold and X-ray crystallographic data are hereby described.
Assuntos
Desenho de Fármacos , Modelos Moleculares , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Piridinas/síntese química , Triazóis/síntese química , Ativação Enzimática/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Piridinas/química , Piridinas/farmacologia , Triazóis/química , Triazóis/farmacologiaRESUMO
Phosphoinositide-3-kinases (PI3K) are a family of lipid kinases mediating numerous cell processes such as proliferation, migration and differentiation. PI3K is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the rapid identification of ETP-46992, within 2-aminocarbonyl imidazo [1,2-a] pyrazine series, with suitable pharmacokinetic (PK) properties that allows the establishment of mechanism of action and efficacy in vivo studies. ETP-46992 showed tumor growth inhibition in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation and in tumor xenograft models with PI3K pathway deregulated (BT474).
Assuntos
Imidazóis/química , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Pirazinas/química , Serina-Treonina Quinases TOR/antagonistas & inibidores , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Citocromos/metabolismo , Modelos Animais de Doenças , Meia-Vida , Humanos , Imidazóis/síntese química , Imidazóis/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Pirazinas/síntese química , Pirazinas/farmacocinética , Serina-Treonina Quinases TOR/metabolismo , Transplante HeterólogoRESUMO
Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the optimization of imidazo [1,2-a] pyrazines, which allow us to identify compound 14 (ETP-46321), with potent biochemical and cellular activity and good pharmacokinetic properties (PK) after oral dosing. ETP-46321 PK/PD studies showed time dependent downregulation of AKT(Ser473) phosphorylation, which correlates with compound levels in tumor tissue and demonstrating to be efficacious in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation. Treatment with ETP-46321 resulted in significant tumor growth inhibition.
Assuntos
Imidazóis/farmacologia , Isoenzimas/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Administração Oral , Disponibilidade Biológica , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacocinética , Tomografia por Emissão de Pósitrons , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Pirazinas/administração & dosagem , Pirazinas/farmacocinética , Tomografia Computadorizada por Raios XRESUMO
This article presents a tool called GDPRValidator that aims to assist small and medium-sized enterprises (SMEs) that have migrated their services, or a part of them, to the cloud to be General Data Protection Regulation (GDPR) compliant when they manage and store employees' or customers' data in the cloud. As these companies have a limited budget to hire legal experts to guide them in complying with GDPR, the main objective of this tool is to help SMEs to be more competitive by saving a considerable amount of money. By using GDPRValidator, these companies can learn and begin the GDPR compliance process by themselves and decide whether it will be necessary to hire GDPR legal experts in the end. GDPRValidator implements a process that aids companies in compliance analysis and validation and generates a series of documents with recommendations. These documents do not guarantee full GDPR compliance, but they can help the company better understand the regulation and improve its data management strategies. In order to validate the efficiency and efficacy of the tool, two SMEs have used it and provided feedback about its perceived ease of use and its perceived usefulness for understanding and complying with GDPR. The results of the validation showed that, for both companies, the degree of perceived usefulness and ease of use of GDPRValidator is quite good. All the scores expressed agreement.
RESUMO
Accurate prediction of human clearance (CL) and volume of distribution at steady state (Vd,ss) for small molecule drug candidates is an essential component of assessing likely efficacious dose and clinical safety margins. In 2021, the IQ Consortium Human PK Prediction Working Group undertook a survey of IQ member companies to understand the current PK prediction methods being used to estimate these parameters across the pharmaceutical industry. The survey revealed a heterogeneity in approaches being used across the industry (e.g., the use of allometric approaches, differing incorporation of binding terms, and inconsistent use of empirical correction factors for in vitro-in vivo extrapolation, IVIVE), which could lead to different PK predictions with the same input data. Member companies expressed an interest in improving human PK predictions by identifying the most appropriate compound-class specific methods, as determined by physiochemical properties and knowledge of CL pathways. Furthermore, there was consensus that increased understanding of the uncertainty inherent to the compound class-dependent prediction would be invaluable in aiding communication of human PK and dose uncertainty at the time of candidate nomination for development. The human PK Prediction Working Group is utilizing these survey findings to help interrogate clinical IV datasets from across the IQ consortium member companies to understand PK prediction accuracy and uncertainty from preclinical datasets.
Assuntos
Indústria Farmacêutica , Modelos Biológicos , Humanos , Cinética , Preparações FarmacêuticasRESUMO
Activation of the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway occurs frequently in a wide range of human cancers and is a main driver of cell growth, proliferation, survival, and chemoresistance of cancer cells. Compounds targeting this pathway are under active development as anticancer therapeutics and some of them have reached advanced clinical trials or been approved by the FDA. Dual PI3K/mTOR inhibitors combine multiple therapeutic efficacies in a single molecule by inhibiting the pathway both upstream and downstream of AKT. Herein, we report our efforts on the exploration of novel small molecule macrocycles (MCXs) as dual PI3K/mTOR inhibitors. Macrocyclization is an attractive approach used in drug discovery, as the semi-rigid character of these structures could provide improved potency, selectivity and favorable pharmacokinetic properties. Importantly, this strategy allows access to new chemical space thus obtaining a better intellectual property position. A series of MCXs based on GSK-2126458, a known clinical PI3K/mTOR inhibitor is described. These molecules showed potent biochemical and cellular dual PI3K/mTOR inhibition, demonstrated strong antitumoral effects in human cancer cell lines, and displayed good drug-like properties. Among them, MCX 83 presented remarkable selectivity against a panel of 468 kinases, high in vitro metabolic stability, and favorable pharmacokinetic parameters without significant CYP450 and h-ERG binding inhibition. This profile qualified this compound as a suitable candidate for future in vivo PK-PD and efficacy studies in mouse cancer models.
Assuntos
Fosfatidilinositol 3-Quinases/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/uso terapêutico , Sulfonamidas/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridazinas , Quinolinas/farmacologia , Sulfonamidas/farmacologiaRESUMO
The PI3K/AKT/mTOR and PIM kinase pathways contribute to the development of several hallmarks of cancer. Cotargeting of these pathways has exhibited promising synergistic therapeutic effects in liquid and solid tumor types. To identify molecules with combined activities, we cross-screened our collection of PI3K/(±mTOR) macrocycles (MCXs) and identified the MCX thieno[3,2-d]pyrimidine derivative 2 as a moderate dual PI3K/PIM-1 inhibitor. We report the medicinal chemistry exploration and biological characterization of a series of thieno[3,2-d]pyrimidine MCXs, which led to the discovery of IBL-302 (31), a potent, selective, and orally bioavailable triple PI3K/mTOR/PIM inhibitor. IBL-302, currently in late preclinical development (AUM302), has recently demonstrated efficacy in neuroblastoma and breast cancer xenografts. Additionally, during the course of our experiments, we observed that macrocyclization was essential to obtain the desired multitarget profile. As a matter of example, the open precursors 35-37 were inactive against PIM whereas MCX 28 displayed low nanomolar activity.
RESUMO
PIM kinase family (PIM-1, PIM-2 and PIM-3) is an appealing target for the discovery and development of selective inhibitors, useful in various disease conditions in which these proteins are highly expressed, such as cancer. The significant effort put, in the recent years, towards the development of small molecules exhibiting inhibitory activity against this protein family has ended up with several molecules entering clinical trials. As part of our ongoing exploration for potential drug candidates that exhibit affinity towards this protein family, we have generated a novel chemical series of triazolo[4,3-b]pyridazine based tricycles by applying a scaffold hopping strategy over our previously reported potent pan-PIM inhibitor ETP-47453 (compound 2). The structure-activity relationship studies presented herein demonstrate a rather selective PIM-1/PIM-3 biochemical profile for this novel series of tricycles, although pan-PIM and PIM-1 inhibitors have also been identified. Selected examples show significant inhibition of the phosphorylation of BAD protein in a cell-based assay. Moreover, optimized and highly selective compounds, such as 42, did not show significant hERG inhibition at 20⯵M concentration, and proved its antiproliferative activity and utility in combination with particular antitumoral agents in several tumor cell lines.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Piridazinas/farmacologia , Quinolinas/farmacologia , Triazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Piridazinas/síntese química , Piridazinas/química , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
Objetivos: Analizar el estigma asociado a la enfermedad mental. Metodología: Se realizó una revisión integradora de carácter descriptivo, realizando una búsqueda bibliográfica utilizando palabras clave relacionadas con el estigma social, el estigma personal y la enfermedad mental. Resultados: Tras la aplicación de los criterios de inclusión establecidos, se seleccionaron y estudiaron un total de 21 artículos. Conclusiones: La literatura consultada muestra que el estigma hacia los trastornos mentales sigue siendo un problema en la sociedad actual, y que acarrea graves consecuencias tanto para los pacientes como para sus familiares (AU)
Objectives: To analyze the stigma associated with mental illness. Methodology: An integrative descriptive review was carried out. The bibliographic search was accomplished using keywords related to social stigma, personal stigma and mental illness. Results: After applying the established inclusion criteria, a total of 21 articles were selected and studied. Conclusions: The spoted literature shows that stigma towards mental disorders continues to be a problem in todays society, and that it has serious consequences for both patients and their families (AU)
Assuntos
Humanos , Idoso , Estigma Social , Transtornos Mentais/psicologia , Autoimagem , EstereotipagemRESUMO
Genetic inhibition of PI3K signaling increases energy expenditure, protects from obesity and metabolic syndrome, and extends longevity. Here, we show that two pharmacological inhibitors of PI3K, CNIO-PI3Ki and GDC-0941, decrease the adiposity of obese mice without affecting their lean mass. Long-term treatment of obese mice with low doses of CNIO-PI3Ki reduces body weight until reaching a balance that is stable for months as long as the treatment continues. CNIO-PI3Ki treatment also ameliorates liver steatosis and decreases glucose serum levels. The above observations have been recapitulated in independent laboratories and using different oral formulations of CNIO-PI3Ki. Finally, daily oral treatment of obese rhesus monkeys for 3 months with low doses of CNIO-PI3Ki decreased their adiposity and lowered their serum glucose levels, in the absence of detectable toxicities. Therefore, pharmacological inhibition of PI3K is an effective and safe anti-obesity intervention that could reverse the negative effects of metabolic syndrome in humans.
Assuntos
Adiposidade/efeitos dos fármacos , Imidazóis/farmacologia , Indazóis/farmacologia , Síndrome Metabólica/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Pirazinas/farmacologia , Sulfonamidas/farmacologia , Adiposidade/fisiologia , Animais , Técnicas Histológicas , Immunoblotting , Fígado/efeitos dos fármacos , Fígado/patologia , Macaca mulatta , Espectrometria de Massas , Camundongos ObesosRESUMO
European Union (EU) agricultural policy has been developed in the pursuit of laudable goals such as a competitive economy and regulatory harmony across the union. However, what has emerged is a fragmented, contradictory, and unworkable legislative framework that threatens economic disaster. In this review, we present case studies highlighting differences in the regulations applied to foods grown in EU countries and identical imported products, which show that the EU is undermining its own competitiveness in the agricultural sector, damaging both the EU and its humanitarian activities in the developing world. We recommend the adoption of rational, science-based principles for the harmonization of agricultural policies to prevent economic decline and lower standards of living across the continent.
Assuntos
Agricultura/legislação & jurisprudência , Produtos Agrícolas , Política Ambiental , União Europeia , Plantas Geneticamente Modificadas , Política Ambiental/economia , Política Ambiental/legislação & jurisprudência , Política Ambiental/tendências , Abastecimento de Alimentos/economia , Abastecimento de Alimentos/legislação & jurisprudência , Regulamentação GovernamentalRESUMO
Nodal and Activin belong to the TGF-ß superfamily and are important regulators of embryonic stem cell fate. Here we investigated whether Nodal and Activin regulate self-renewal of pancreatic cancer stem cells. Nodal and Activin were hardly detectable in more differentiated pancreatic cancer cells, while cancer stem cells and stroma-derived pancreatic stellate cells markedly overexpressed Nodal and Activin, but not TGF-ß. Knockdown or pharmacological inhibition of the Nodal/Activin receptor Alk4/7 in cancer stem cells virtually abrogated their self-renewal capacity and in vivo tumorigenicity, and reversed the resistance of orthotopically engrafted cancer stem cells to gemcitabine. However, engrafted primary human pancreatic cancer tissue with a substantial stroma showed no response due to limited drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor enhanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free survival. Therefore, inhibition of the Alk4/7 pathway, if combined with hedgehog pathway inhibition and gemcitabine, provides a therapeutic strategy for targeting cancer stem cells.