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Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in understanding of the pharmacokinetics/pharmacodynamics (PK/PD) and disposition of this modality exist. This position paper comes from the Novel Modalities Working Group (WG), part of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). The pan-industry WG effort focuses on the nonclinical PK and clinical pharmacology aspects of AAV gene therapy and related bioanalytical considerations.Traditional PK concepts are generally not applicable to AAV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction and transgene-derived protein expression. Therefore, we explain PK concepts of biodistribution of AAV-based therapies and place key terminologies related to drug exposure and PD in the proper context. Factors affecting biodistribution are presented in detail, and guidelines are provided to design nonclinical studies to enable a stage-gated progression to Phase 1 testing. The nonclinical and clinical utility of transgene DNA, mRNA, and protein analytes are discussed with bioanalytical strategies to measure these analytes. The pros and cons of qPCR vs. ddPCR technologies for DNA/RNA measurement and qualitative vs. quantitative methods for transgene-derived protein are also presented. Last, best practices and recommendations for use of clinical and nonclinical data to project human dose and response are discussed. Together, the manuscript provides a holistic framework to discuss evolving concepts of PK/PD modeling, bioanalytical technologies, and clinical dose selection in gene therapy.
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Dependovirus , Terapia Genética , Humanos , Dependovirus/genética , Distribución Tisular , Desarrollo de Medicamentos , Reacción en Cadena de la PolimerasaRESUMEN
Immunogenicity has imposed a challenge to efficacy and safety evaluation of adeno-associated virus (AAV) vector-based gene therapies. Mild to severe adverse events observed in clinical development have been implicated with host immune responses against AAV gene therapies, resulting in comprehensive evaluation of immunogenicity during nonclinical and clinical studies mandated by health authorities. Immunogenicity of AAV gene therapies is complex due to the number of risk factors associated with product components and pre-existing immunity in human subjects. Different clinical mitigation strategies have been employed to alleviate treatment-induced or -boosted immunogenicity in order to achieve desired efficacy, reduce toxicity, or treat more patients who are seropositive to AAV vectors. In this review, the immunogenicity risk assessment, manifestation of immunogenicity and its impact in nonclinical and clinical studies, and various clinical mitigation strategies are summarized. Last, we present bioanalytical strategies, methodologies, and assay validation applied to appropriately monitor immunogenicity in AAV gene therapy-treated subjects.
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The liver is central to the elimination of many drugs from the body involving multiple processes and understanding of these processes is important to quantitively assess hepatic clearance of drugs. The synthetic STING (STimulator of INterferon Genes protein) agonist is a new class of drugs currently being evaluated in clinical trials as a potential anticancer therapy. In this study, we used ML00960317 (synthetic STING agonist) to investigate the hepatobiliary disposition of this novel molecular entity. A bile-duct cannulated (BDC) rat study indicated that biliary excretion is the major route of elimination for ML00960317 (84% of parent dose in bile). The human biliary clearance using in vitro sandwich cultured human hepatocyte model predicted significant biliary excretion of ML00960317 (biliary excretion index (BEI) of 47%). Moreover, the transport studies using transporter expressing cell lines, hepatocytes, and membrane vesicles indicated that ML00960317 is a robust substrate of OATP1B1, OATP1B3, and MRP2. Using relative expression factor approach, the combined contribution of OATP1B1 (fraction transported (ft) = 0.62) and OATP1B3 (ft = 0.31) was found to be 93% of the active uptake clearance of ML00960317 into the liver. Furthermore, OATP1B1 and OATP1B3-mediated uptake of ML00960317 was inhibited by rifampicin with IC50 of 6.5 and 2.3 µM, respectively indicating an in vivo DDI risk (R value of 1.5 and 2.5 for OATP1B1 and OATP1B3, respectively). These results highlighted an important role of OATP1B1, OATP1B3, and MRP2 in the hepatobiliary disposition of ML00960317. These pathways may act as rate-determining steps in the hepatic clearance of ML00960317 thus presenting clinical DDI risk.
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Bilis , Transportadores de Anión Orgánico , Animales , Aniones/metabolismo , Bilis/metabolismo , Humanos , Interferones/metabolismo , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Transportadores de Anión Orgánico/metabolismo , Péptidos , Ratas , Rifampin , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones OrgánicosRESUMEN
Chimeric antigen receptor (CAR) T cell therapies have revolutionized the treatment of hematologic malignancies and have potentials for solid tumor treatment. To overcome limited CAR T cell infiltration to solid tumors, local delivery of CAR T cells is a practical strategy that has shown promising therapeutic outcome and safety profile in the clinic. It is of great interest to understand the impact of dosing routes on CAR T cell distribution, subsequent proliferation and tumor killing in a quantitative manner to identify key factors that contribute to CAR T efficacy and safety. In this study, we established mouse minimal physiologically-based pharmacokinetic (mPBPK) models combined with pharmacodynamic (PD) components to delineate CAR T cell distribution, proliferation, tumor growth, and tumor cell killing in the cases of pleural and liver tumors. The pleural tumor model reasonably captured published CAR T cellular kinetic and tumor growth profiles in mice. The mPBPK-PD simulation of a liver tumor mouse model showed a substantial increase in initial tumor infiltration and earlier CAR T cell proliferation with local hepatic artery delivery compared to portal vein and intravenous (i.v.) injections whereas portal vein injection showed little difference from i.v. administration, suggesting the importance of having the injection site close to tumor for maximal effect of non-systemic administration. Blood flow rate in the liver tumor was found to be a sensitive parameter for cellular kinetics and efficacy, indicating a potential role of tumor vascularization in the efficacy of CAR T cell therapies.
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Neoplasias Hepáticas , Receptores Quiméricos de Antígenos , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Inmunoterapia Adoptiva , Ratones , Linfocitos TRESUMEN
Drug metabolism and pharmacokinetics (DMPK) is an important branch of pharmaceutical sciences. The nature of ADME (absorption, distribution, metabolism, excretion) and PK (pharmacokinetics) inquiries during drug discovery and development has evolved in recent years from being largely descriptive to seeking a more quantitative and mechanistic understanding of the fate of drug candidates in biological systems. Tremendous progress has been made in the past decade, not only in the characterization of physiochemical properties of drugs that influence their ADME, target organ exposure, and toxicity, but also in the identification of design principles that can minimize drug-drug interaction (DDI) potentials and reduce the attritions. The importance of membrane transporters in drug disposition, efficacy, and safety, as well as the interplay with metabolic processes, has been increasingly recognized. Dramatic increases in investments on new modalities beyond traditional small and large molecule drugs, such as peptides, oligonucleotides, and antibody-drug conjugates, necessitated further innovations in bioanalytical and experimental tools for the characterization of their ADME properties. In this review, we highlight some of the most notable advances in the last decade, and provide future perspectives on potential major breakthroughs and innovations in the translation of DMPK science in various stages of drug discovery and development.
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The correlation between efficacious doses in human tumor-xenograft mouse models and the human clinical doses of approved oncology agents was assessed using published preclinical data and recommended clinical doses. For 90 approved small molecule anti-cancer drugs, body surface area (BSA) corrected mouse efficacious doses were strongly predictive of human clinical dose ranges with 85.6% of the predictions falling within three-fold (3×) of the recommended clinical doses and 63.3% within 2×. These results suggest that BSA conversion is a useful tool for estimating human doses of small molecule oncology agents from mouse xenograft models from the early discovery stage. However, the BSA based dose conversion poorly predicts for the intravenous antibody and antibody drug conjugate anti-cancer drugs. For antibody-based drugs, five out of 30 (16.7%) predicted doses were within 3× of the recommended clinical dose. The body weight-based dose projection was modestly predictive with 66.7% of drugs predicted within 3× of the recommended clinical dose. The correlation was slightly better in ADCs (77.7% in 3×). The application and limitations of such simple dose estimation methods in the early discovery stage and in the design of clinical trials are also discussed in this retrospective analysis.
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Given the recent success of gene therapy modalities and the growing number of cell and gene-based therapies in clinical development across many different therapeutic areas, it is evident that this evolving field holds great promise for the unmet medical needs of patients. The recent approvals of Luxturna® and Zolgensma® prove that recombinant adeno-associated virus (rAAV)-based gene therapy is a transformative modality that enables curative treatment for genetic disorders. Over the last decade, Takeda has accumulated significant experience with rAAV-based gene therapies, especially in the early stage of development. In this review, based on the learnings from Takeda and publicly available information, we aim to provide a guiding perspective on Drug Metabolism and Pharmacokinetics (DMPK) substantial role in advancing therapeutic gene therapy modalities from nonclinical research to clinical development, in particular the characterization of gene therapy product biodistribution, elimination (shedding), immunogenicity assessment, multiple platform bioanalytical assays, and first-in-human (FIH) dose projection strategies. Graphical abstract.
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Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Animales , Productos Biológicos , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/terapia , Humanos , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody-dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).
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Inmunoconjugados/uso terapéutico , Animales , Femenino , Células HEK293 , Humanos , Inmunoconjugados/farmacología , Ratones , Ratones Desnudos , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ectoenzyme CD38 is increased on lymphocytes in response to an antigenic challenge and it is hypothesized that targeting these activated lymphocytes could ameliorate pathologic activities in autoimmune diseases. The cynomolgus monkey is an appropriate model for assessing potential effects of targeting CD38 in humans because these species exhibit similar expression profiles. TAK-079 is a human monoclonal antibody (IgG1 λ ) that binds to CD38 and lyses bound cells by complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. TAK-079 binds to monkey CD38 with an affinity at EC50 4.5 nM, and the potential activity of TAK-079 was investigated in a monkey collagen-induced arthritis model of autoimmune disease. Prophylactic administration of TAK-079 (3 mg/kg i.v. weekly) was well tolerated and prevented arthritis development compared with vehicle-treated control animals, which exhibited progressive disease with radiographic damage and worsening clinical scores over the study course. Therapeutic treatment of arthritic monkeys with TAK-079 (3 mg/kg i.v. weekly) was also well tolerated and reduced disease progression and symptoms. Arthritis scores and joint swelling were significantly lower than the vehicle control, accompanied by decreases in blood levels of C-reactive protein, alkaline phosphatase, and natural killer, B, and T cells. Histopathology, morphometry, and radiology revealed significantly less joint damage in animals exposed prophylactically to TAK-079 treatment compared with vehicle-treated animals and significantly less damage in animals treated therapeutically with TAK-079 or dexamethasone (0.1 mg/kg oral gavage daily), illustrating potential disease-modifying activity. In conclusion, these data indicate that depletion of CD38-expressing cells could be a therapeutic mechanism for treating autoimmune diseases. SIGNIFICANCE STATEMENT: This study demonstrates that targeting CD38-expressing leukocytes with a cytolytic antibody can ameliorate autoimmune disease in cynomolgus monkeys. The study gives a unique perspective into this therapeutic strategy because the three other anti-CD38 cytolytic antibodies in clinical development (daratumumab, isatuximab, and MOR202) cannot be tested in similar models because they do not crossreact with CD38 expressed by new world primates.
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ADP-Ribosil Ciclasa 1/metabolismo , Anticuerpos Monoclonales/inmunología , Artritis Experimental/inmunología , Linfocitos B/metabolismo , Regulación de la Expresión Génica/inmunología , Células Asesinas Naturales/metabolismo , Linfocitos T/metabolismo , ADP-Ribosil Ciclasa 1/inmunología , Animales , Artritis Experimental/metabolismo , Artritis Experimental/patología , Linfocitos B/inmunología , Células CHO , Cricetulus , Progresión de la Enfermedad , Células Asesinas Naturales/inmunología , Macaca fascicularis , Linfocitos T/inmunologíaRESUMEN
A thorough understanding of species-dependent differences in hepatic uptake transporters is critical for predicting human pharmacokinetics (PKs) from preclinical data. In this study, the activities of organic anion transporting polypeptide (OATP/Oatp), organic cation transporter 1 (OCT1/Oct1), and sodium-taurocholate cotransporting polypeptide (NTCP/Ntcp) in cultured rat, dog, monkey and human hepatocytes were compared. The activities of hepatic uptake transporters were evaluated with respect to culture duration, substrate and species-dependent differences in hepatocytes. Longer culture duration reduced hepatic uptake transporter activities across species except for Oatp and Ntcp in rats. Comparable apparent Michaelis-Menten constant (Km,app) values in hepatocytes were observed across species for atorvastatin, estradiol-17ß-glucuronide and metformin. The Km,app values for rosuvastatin and taurocholate were significantly different across species. Rat hepatocytes exhibited the highest Oatp percentage of uptake transporter-mediated permeation clearance (PSinf,act) while no difference in %PSinf,act of probe substrates were observed across species. The in vitro hepatocyte inhibition data in rats, monkeys and humans provided reasonable predictions of in vivo drug-drug interaction (DDIs) between atorvastatin/rosuvastatin and rifampin. These findings suggested that using human hepatocytes with a short culture time is the most robust preclinical model for predicting DDIs for compounds exhibiting active hepatic uptake in humans.
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Proteínas de Transporte de Catecolaminas en la Membrana Plasmática/metabolismo , Hepatocitos/metabolismo , Modelos Biológicos , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Adulto , Animales , Atorvastatina/farmacocinética , Atorvastatina/farmacología , Transporte Biológico Activo , Estradiol/análogos & derivados , Estradiol/farmacocinética , Estradiol/farmacología , Femenino , Hepatocitos/citología , Humanos , Masculino , Metformina/farmacocinética , Metformina/farmacología , Persona de Mediana Edad , Ratas , Ratas Sprague-DawleyRESUMEN
1. Breast cancer resistance protein (BCRP) plays an important role in drug absorption, distribution and excretion. It is challenging to evaluate BCRP functions in preclinical models because commonly used BCRP inhibitors are nonspecific or unstable in animal plasma. 2. In this work, in vitro absorption, distribution, metabolism and elimination (ADME) assays and pharmacokinetic (PK) experiments in Bcrp knockout (KO) (Abcg2-/-) and wild-type (WT) FVB mice and Wistar rats were conducted to characterize the preclinical properties of a novel selective BCRP inhibitor (ML753286, a Ko143 analog). 3. ML753286 is a potent inhibitor for BCRP, but not for P-glycoprotein (P-gp), organic anion-transporting polypeptide (OATP) or major cytochrome P450s (CYPs). It has high permeability, but is not an efflux transporter substrate. ML753286 has low to medium clearance in rodent and human liver S9 fractions, and is stable in plasma cross species. Bcrp inhibition affects oral absorption and clearance of sulfasalazine in rodents. A single dose of ML753286 at 50-300 mg/kg orally, and at 20 mg/kg intravenously or 25 mg/kg orally inhibits Bcrp functions in mice and rats, respectively. 4. These findings confirm that ML753286 is a useful selective inhibitor to evaluate BCRP/Bcrp activity in vitro and in rodent model systems.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Absorción Fisiológica , Neoplasias de la Mama/tratamiento farmacológico , Dicetopiperazinas/farmacocinética , Dicetopiperazinas/uso terapéutico , Proteínas de Neoplasias/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Células CACO-2 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Dicetopiperazinas/sangre , Dicetopiperazinas/química , Perros , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Macaca fascicularis , Masculino , Ratones Noqueados , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Ratas , Sulfasalazina/farmacología , Sulfasalazina/uso terapéutico , Factores de TiempoRESUMEN
1. Red blood cell (RBC) partitioning is important in determining pharmacokinetic and pharmacodynamic properties of a compound; however, active transport across RBC membranes is not well understood, particularly without transporter-related cell membrane proteomics data. 2. In this study, we quantified breast cancer resistance protein (BCRP/Bcrp) and MDR1/P-glycoprotein (P-gp) protein expression in RBCs from humans, monkeys, dogs, rats and mice using nanoLC/MS/MS, and evaluated their effect on RBC partitioning and plasma exposure of their substrates. BCRP-specific substrate Cpd-1 and MDR1-specific substrate Cpd-2 were characterized using Caco-2 Transwell® system and then administered to Bcrp or P-gp knockout mice. 3. The quantification revealed BCRP/Bcrp but not MDR1/P-gp to be highly expressed on RBC membranes. The knockout mouse study indicated BCRP/Bcrp pumps the substrate out of RBCs, lowering its partitioning and thus preventing binding to intracellular targets. This result was supported by a Cpd-1 and Bcrp inhibitor ML753286 drug-drug interaction (DDI) study in mice. Because of enhanced partitioning of Cpd-1 into RBCs after BCRP/Bcrp inhibition, Cpd-1 plasma concentration changed much less extent with genetic or chemical knockout of Bcrp albeit marked blood concentration increase, suggesting less DDI effect. 4. This finding is fundamentally meaningful to RBC partitioning, pharmacokinetics and DDI studies of BCRP-specific substrates.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas de Neoplasias/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Animales , Células CACO-2 , Cromatografía Liquida , Interacciones Farmacológicas , Membrana Eritrocítica/efectos de los fármacos , Femenino , Humanos , Macaca fascicularis , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas de Neoplasias/antagonistas & inhibidores , Ratas , Espectrometría de Masas en Tándem , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND AND OBJECTIVES: Monomethyl auristatin E (MMAE), the toxin linked to CD30-specific monoclonal antibody of Adcetris® (brentuximab vedotin), is a potent anti-microtubule agent. Brentuximab vedotin has been approved for the treatment of relapsed or refractory Hodgkin lymphoma and anaplastic large cell lymphoma. Cytochrome P450 (CYP) induction assessment of MMAE was conducted in human hepatocytes to assess DDI potentials and its translation to clinic. METHODS: MMAE was incubated at 1-1000 nM with cultured primary human hepatocytes for 72 h, and CYP1A2, CYP2B6, and CYP3A4 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction and CYP-specific probe substrate by liquid chromatography coupled with mass spectrometry, along with microtubule disruption by immunofluorescence staining using anti-ß-tubulin antibody and imaging. RESULTS: MMAE up to 10 nM had no significant effect on CYP1A2, CYP2B6, and CYP3A4 mRNA expression and activity, whereas at higher concentrations of 100- and 1000-nM MMAE, the CYP mRNA expression and activity were diminished substantially. Further investigation showed that the degree of CYP suppression was paralleled by that of microtubule disruption by MMAE, as measured by increase in the number of ß-tubulin-positive aggregates. At the clinical dose, the concentration of MMAE was 7 nM which did not show any significant CYP suppression or microtubule disruption in hepatocytes. CONCLUSIONS: MMAE was not a CYP inducer in human hepatocytes. However, it caused a concentration-dependent CYP mRNA suppression and activity. The CYP suppression was associated with microtubule disruption, supporting the reports that intact microtubule architecture is required for CYP regulations. The absence of CYP suppression and microtubule disruption in vitro at the clinical plasma concentrations of MMAE (< 10 nM) explains the lack of pharmacokinetic drug interaction between brentuximab vedotin and midazolam, a sensitive CYP3A substrate, reported in patients.
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Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Inmunoconjugados/farmacología , Microtúbulos/efectos de los fármacos , Oligopéptidos/farmacología , Antineoplásicos/farmacología , Brentuximab Vedotina , Células Cultivadas , Interacciones Farmacológicas , Humanos , Microtúbulos/metabolismo , ARN Mensajero/metabolismoRESUMEN
The intracellular unbound inhibitor concentration ([I]unbound,cell) is the most relevant concentration for predicting the inhibition of hepatic efflux transporters. However, the intracellular unbound fraction of inhibitor in hepatocytes (fu,cell,inhibitor) is not routinely determined. Studies are needed to evaluate the benefit of measuring fu,cell,inhibitor and using [I]unbound,cell versus intracellular total inhibitor concentration ([I]total,cell) when predicting inhibitory effects. This study examined the benefit of using [I]unbound,cell to predict hepatocellular bile acid disposition. Cellular total concentrations of taurocholate ([TCA]total,cell), a prototypical bile acid, were simulated using pharmacokinetic parameters estimated from sandwich-cultured human hepatocytes. The effect of various theoretical inhibitors was simulated by varying ([I]total,cell/ half maximal inhibitory concentration [IC50]) values. In addition, the fold change was calculated as the simulated [TCA]total,cell when fu,cell,inhibitor = 1 divided by the simulated [TCA]total,cell when fu,cell,inhibitor = 0.5-0.01. The lowest ([I]total,cell/IC50) value leading to a >2-fold change in [TCA]total,cell was chosen as a cutoff, and a framework was developed to categorize risk inhibitors for which the measurement of fu,cell,inhibitor is optimal. Fifteen compounds were categorized, 5 of which were compared with experimental observations. Future work is needed to evaluate this framework based on additional experimental data. In conclusion, the benefit of measuring fu,cell,inhibitor to predict hepatic efflux transporter-mediated drug-bile acid interactions can be determined a priori.
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Ácidos y Sales Biliares/metabolismo , Hepatocitos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Preparaciones Farmacéuticas/metabolismo , Transporte Biológico/efectos de los fármacos , Simulación por Computador , Interacciones Farmacológicas , Hepatocitos/efectos de los fármacos , Humanos , Modelos Biológicos , Unión Proteica , Ácido Taurocólico/metabolismoRESUMEN
Drug-induced liver injury (DILI) is a severe drug adverse response, which cannot always be reliably predicted in preclinical or clinical studies. Lack of observation of DILI during preclinical and clinical drug development has led to DILI being a leading cause of drug withdrawal from the market. As DILI is potentially fatal, pharmaceutical companies have been developing in vitro tools to screen for potential liver injury. Screens for physicochemical properties, mitochondrial function, and transport protein inhibition have all been employed to varying degrees of success. In vitro inhibition of the bile salt export pump (BSEP) has become a major risk factor for in vivo DILI predictions, yet discrepancies exist in which methods to use and the extent to which BSEP inhibition predicts clinical DILI. The presented work focuses on optimizing DILI predictions by comparing BSEP inhibition via the membrane vesicle assay and the hepatocyte-based BSEPcyte assay, as well as dual and triple liabilities. BSEP transport inhibition of taurcholic acids and glycocholic acids were similar for up to 29 drugs tested, in both the vesicle and hepatocyte-based assays. Positive and negative DILI predictions were optimized at a 50-µM cutoff value for 50 drugs using both NIH Livertox and PharmaPendium databases. Additionally, dual inhibition of BSEP and other efflux transporters (multidrug resistance-associated protein [MRP]2, MRP3, or MRP4) provided no observable predictive benefit compared with BSEP inhibition alone. Eighty-five percent of drugs with high molecular weight (>600 Da), high cLogP (>3), or a daily dose >100 mg and BSEP inhibition were associated with DILI. Triple liability of BSEP inhibition, high molecular weight, and high cLogP attained a 100% positive prediction rate.
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Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/antagonistas & inhibidores , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Hepatocitos/efectos de los fármacos , Preparaciones Farmacéuticas/química , Células Cultivadas , Fenómenos Químicos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Hepatocitos/metabolismo , Humanos , Concentración 50 Inhibidora , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Valor Predictivo de las Pruebas , Factores de RiesgoRESUMEN
For many orally administered basic drugs with pH-dependent solubility, concurrent administration with acid-reducing agents (ARAs) can significantly impair their absorption and exposure. In this study, pH-dependent drug-drug interaction (DDI) prediction methods, including in vitro dissolution-permeation chamber (IVDP) and physiologically based pharmacokinetic (PBPK) modeling, were evaluated for their ability to quantitatively predict the clinical DDI observations using 11 drugs with known clinical pH-dependent DDI data. The data generated by IVDP, which consists of a gastrointestinal compartment and a systemic compartment separated by a biomimic membrane, significantly correlated with the clinical DDI observations. The gastrointestinal compartment AUC ratio showed strong correlation with clinical AUC ratio (R=0.72 and P=0.0056), and systemic compartment AUC ratio showed strong correlation with clinical Cmax ratio (R=0.91 and P=0.0003). PBPK models were also developed for the 11 test compounds. The simulations showed that the predictions from PBPK model with experimentally measured parameters significantly correlated with the clinical DDI observations. Future studies are needed to evaluate predictability of Z-factor-based PBPK models for pH-dependent DDI. Overall, these data suggested that the severity of pH-dependent DDI can be predicted by in vitro and in silico methods. Proper utilization of these methods before clinical DDI studies could allow adequate anticipation of pH-dependent DDI, which helps with minimizing pharmacokinetic variation in clinical studies and ensuring every patient with life-threatening diseases receives full benefit of the therapy.
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Modelos Teóricos , Farmacocinética , Interacciones Farmacológicas , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Permeabilidad , SolubilidadRESUMEN
PURPOSE: The expression levels of several efflux drug transporters in the liver and kidney were evaluated across species to address potential roles of the transporters in species dependent excretion of drugs and their metabolites. METHODS: Four efflux transporters, namely MDR1/P-gp, BCRP/Bcrp, MRP2/Mrp2 and MRP3/Mrp3 in liver and kidney in three preclinical species and humans were quantified using targeted quantitative proteomics by isotope dilution nanoLC-MS/MS. RESULTS: In liver, the level of P-gp was highest in monkey and lowest in rat. The concentration of BCRP/Bcrp was highest in dog followed by monkey. MRP2/Mrp2 level was highest in monkey and rat, whereas MRP3/Mrp3 levels were similar in human, monkey and dog. In the kidney, the concentrations of MDR1/P-gp in human and monkey were roughly 2 to 3-fold higher than in rat and dog. In rat, BCRP/Bcrp concentrations were substantially higher than in any of the other species. MRP2/Mrp2 concentrations were similar across species, whereas expression of MRP3/Mrp3 was highest in rat. CONCLUSION: Overall, the results indicated that the pattern of hepatic and renal expression of the transporters was quite species dependent. This information should be helpful in the estimation of transport mediated drug and metabolites excretion in liver and kidney across species.
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Isótopos/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Preparaciones Farmacéuticas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico/fisiología , Perros , Femenino , Haplorrinos , Humanos , Proteínas de Transporte de Membrana/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteómica/métodos , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem/métodosRESUMEN
The design, synthesis, in vitro inhibitory potency, and pharmacokinetic (PK) profiles of Ko143 analogs are described. Compared to commonly used Ko143, the new breast cancer resistance protein (BCRP) inhibitor (compound A) showed the same potency and a significantly improved PK profile in rats (lower clearance [1.54L/h/kg] and higher bioavailability [123%]). Ko143 on the other hand suffers from poor bioavailability. Compared to Ko143, compound A would be a useful probe for delineating the role of BCRP during in vivo studies in animals.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Dicetopiperazinas/síntesis química , Dicetopiperazinas/farmacocinética , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Animales , Células CACO-2 , Estrona/análogos & derivados , Estrona/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/sangre , Humanos , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Human hepatic organic anion-transporting polypeptides (OATPs), including OATP1B1, 1B3, and 2B1, are expressed at the basolateral membrane of hepatocytes and mediate the uptake of a variety of compounds from blood into hepatocytes. The liver-specific OATPs are increasingly recognized as playing important roles in the pharmacokinetic (PK) of many drugs and thus, involved in the clinically significant drug-drug interactions (DDIs). However, the evaluation of the specific roles of individual OATPs in hepatocytes is challenging because of the lack of selective inhibitors and probe substrates for each OATP member. In the present study, the uptake activity of OATP1B3 was examined in human hepatocytes cultured up to 14 days using an in vitro uptake assay. The results showed that OATP-mediated uptake of rosuvastatin, a substrate for OATPs declined substantially in cultured human hepatocytes. In contrast, the uptake of OATP1B3-selective substrate telmisartan was not measureable at earlier culture periods, but became detectable on Day 7 and showed culture duration-dependent changes from Day 7 to 14. Quantitative polymerase chain reaction (qPCR) analyses illustrated that the OATP functional change was not correlated with messenger ribonucleic acid (mRNA) expression alteration in hepatocytes cultured for 3 hours or 7 days. The OATP1B3-mediated telmisartan uptake was also culture medium- and donor-dependent, and only observed in 3 of 5 lots of hepatocytes cultured in 2 of 3 media tested. These results show that using human hepatocytes cultured in certain conditions may provide an excellent addition to transfected cell lines as a way to distinguish OATP1B3 from other hepatic OATP family members, such as OATP1B1, to provide more understanding of OATP-mediated clinical DDI.
Asunto(s)
Bencimidazoles/metabolismo , Benzoatos/metabolismo , Técnicas de Cultivo de Célula , Hepatocitos/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Donantes de Tejidos , Anciano , Transporte Biológico , Medios de Cultivo/metabolismo , Femenino , Fluorobencenos/metabolismo , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Transportadores de Anión Orgánico Sodio-Independiente/genética , Pirimidinas/metabolismo , ARN Mensajero/metabolismo , Rosuvastatina Cálcica , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos , Sulfonamidas/metabolismo , Telmisartán , Factores de Tiempo , Transfección , Adulto JovenRESUMEN
Alisertib (MLN8237) is an investigational potent Aurora A kinase inhibitor currently under clinical trials for hematological and nonhematological malignancies. Nonclinical investigation showed that alisertib is a highly permeable compound with high plasma protein binding, low plasma clearance, and moderate volume of distribution in rats, dogs, monkeys and chimpanzees. Consistent with the above properties, the oral bioavailability in animals was greater than 82%. The predicted human oral pharmacokinetic (PK) profile was constructed using allometric scaling of plasma clearance and volume of distribution in the terminal phase from animals. The chimpanzee PK profiles were extremely useful to model absorption rate constant, which was assumed to be similar to that in humans, based on the fact that chimpanzees are phylogenetically closest to humans. The human plasma clearance was projected to be low of 0.12 L/hr/kg, with half-life of approximately 10 hr. For human efficacious dose estimation, the tumor growth inhibition as a measure of efficacy (E) was assessed in HCT116 xenograft mice at several oral QD or BID dose levels. Additionally, subcutaneous mini-pump infusion studies were conducted to assess mitotic index in tumor samples as a pharmacodynamic (PD) marker. PK/PD/E modeling showed that for optimal efficacy and PD in the xenograft mice maintaining a plasma concentration exceeding 1 µM for at least 8-12 hr would be required. These values in conjunction with the projected human PK profile estimated the optimal oral dose of approximately 103 mg QD or 62.4 mg BID in humans. Notably, the recommended Phase 2 dose being pursued in the clinic is close to the projected BID dose.