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The dose dependence of the effect of enzyme inducers and the effect of the combined administration of two inducers that exert their effect via the same induction pathway (pregnane X receptor) have not been well studied. Using oral midazolam microdoses (30 µg), we have investigated CYP3A4 induction by St. John's wort (SJW) in 11 healthy volunteers using low (300 mg/day containing 7.48 mg hyperforin), therapeutic (900 mg/day), and supratherapeutic doses of SJW (1800 mg/day) for 14 days. SJW was then co-administered with rifampin (600 mg/day) for a further 7 days to evaluate the effect of the combined administration of two inducers. In addition, intravenous midazolam microdoses (10 µg) were administered before SJW, at SJW 1800 mg/day, and during administration of the two inducers to assess the hepatic contribution to total induction (semi-simultaneous administration). Administration of SJW increased oral midazolam clearance 1.96-fold (300 mg/day), 3.86-fold (900 mg/day), and 5.62-fold (1800 mg/day), and 17.5-fold after the addition of rifampin. Concurrently, the clearance of intravenous midazolam increased 2.05-fold (1800 mg/day) and 2.93-fold (SJW + rifampin). These results show that rifampin significantly enhances the induction of the highest SJW doses both hepatically and overall and suggest that these metabolic effects occur predominantly in the gut. These findings also suggest that in drug interactions involving strong and moderate enzyme inducers, the perpetrator effects of the strong inducer are decisive for the interaction.
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Inductores del Citocromo P-450 CYP3A , Citocromo P-450 CYP3A , Relación Dosis-Respuesta a Droga , Hypericum , Midazolam , Rifampin , Rifampin/administración & dosificación , Rifampin/farmacología , Humanos , Hypericum/química , Midazolam/administración & dosificación , Midazolam/farmacocinética , Midazolam/farmacología , Citocromo P-450 CYP3A/metabolismo , Masculino , Adulto , Inductores del Citocromo P-450 CYP3A/farmacología , Inductores del Citocromo P-450 CYP3A/administración & dosificación , Femenino , Adulto Joven , Administración Oral , Extractos Vegetales/administración & dosificación , Extractos Vegetales/farmacología , Inducción Enzimática/efectos de los fármacosRESUMEN
Drug efflux transporters of the ATP-binding-cassette superfamily play a major role in the availability and concentration of drugs at their site of action. ABCC2 (MRP2) and ABCG2 (BCRP) are among the most important drug transporters that determine the pharmacokinetics of many drugs and whose overexpression is associated with cancer chemoresistance. ABCC2 and ABCG2 expression is frequently altered during treatment, thus influencing efficacy and toxicity. Currently, there are no routine approaches available to closely monitor transporter expression. Here, we developed and validated a UPLC-MS/MS method to quantify ABCC2 and ABCG2 in extracellular vesicles (EVs) from cell culture and plasma. In this way, an association between ABCC2 protein levels and transporter activity in HepG2 cells treated with rifampicin and hypericin and their derived EVs was observed. Although ABCG2 was detected in MCF7 cell-derived EVs, the transporter levels in the vesicles did not reflect the expression in the cells. An analysis of plasma EVs from healthy volunteers confirmed, for the first time at the protein level, the presence of both transporters in more than half of the samples. Our findings support the potential of analyzing ABC transporters, and especially ABCC2, in EVs to estimate the transporter expression in HepG2 cells.
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Vesículas Extracelulares , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Humanos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Cromatografía Liquida , Proteínas de Neoplasias/genética , Espectrometría de Masas en Tándem , Proteínas de Transporte de MembranaRESUMEN
Cisplatin not only targets DNA but also RNA. However, it is largely unknown whether platinated RNA (Pt-RNA) causes apoptosis and thus contributes to the cytotoxic effects of cisplatin. Consequently, cellular RNA was isolated from HepG2 and LS180 cells, exposed to cisplatin, and the resulting Pt-RNA (20â¯ng Pt/µg RNA) was transfected into these cancer cell lines or used to treat an apoptosis reporter Caenorhabditis elegans (C. elegans) strain (MD701, expressing CED-1::GFP). Cellular and molecular effects of Pt-RNA were evaluated by luminogenic caspase 3/7 assays, PCR array analysis, and fluorescence microscopy-based quantification of apoptosis in C. elegans gonads. Assuming RNA cross-linking (pseudo double-stranded RNA), the contribution of the Toll-like receptor 3 (TLR3, a sensor of double-stranded RNA) to apoptosis induction in cancer cell lines was investigated by pharmacological TLR3 inhibition and overexpression. In contrast to controls, Pt-RNA significantly enhanced apoptosis in C. elegans (2-fold) and in the cancer cell lines (2-fold to 4-fold). TLR3 overexpression significantly enhanced the pro-apoptotic effects of Pt-RNA in HepG2 cells. TLR3 inhibition reduced the pro-apoptotic effects of Pt-RNA and cisplatin, but not of paclitaxel (off-target control). Gene expression analysis showed that Pt-RNA (but not RNA) significantly enhanced the mRNA levels of nuclear factor kappa B subunit 2 and interleukin-8 in HepG2 cells, suggesting that Pt-RNA is a damage-associated molecular pattern that additionally causes pro-inflammatory responses. Together, this data suggests that not only DNA but also cellular RNA is a functionally relevant target of cisplatin, leading to pro-apoptotic and immunogenic effects.
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Cisplatino , Neoplasias , Animales , Cisplatino/farmacología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/farmacología , Apoptosis , Línea Celular Tumoral , ADN , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
BACKGROUND: We investigated the effect of a 5-day low-dose ritonavir therapy, as it is used in the treatment of COVID-19 with nirmatrelvir/ritonavir, on the pharmacokinetics of three factor Xa inhibitors (FXaI). Concurrently, the time course of the activities of the cytochromes P450 (CYP) 3A4, 2C19, and 2D6 was assessed. METHODS: In an open-label, fixed sequence clinical trial, the effect and duration of a 5-day oral ritonavir (100 mg twice daily) treatment on the pharmacokinetics of three oral microdosed FXaI (rivaroxaban 25 µg, apixaban 25 µg, and edoxaban 50 µg) and microdosed probe drugs (midazolam 25 µg, yohimbine 50 µg, and omeprazole 100 µg) was evaluated in eight healthy volunteers. The plasma concentrations of all drugs were quantified using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and pharmacokinetics were analysed using non-compartmental analyses. RESULTS: Ritonavir increased the exposure of apixaban, edoxaban, and rivaroxaban, but to a different extent the observed area under the plasma concentration-time curve (geometric mean ratio 1.29, 1.46, and 1.87, respectively). A strong CYP3A4 inhibition (geometric mean ratio > 10), a moderate CYP2C19 induction 2 days after ritonavir (0.64), and no alteration of CYP2D6 were observed. A CYP3A4 recovery half-life of 2.3 days was determined. CONCLUSION: This trial with three microdosed FXaI suggests that at most the rivaroxaban dose should be reduced during short-term ritonavir, and only in patients receiving high maintenance doses. Thorough time series analyses demonstrated differential effects on three different drug-metabolising enzymes over time with immediate profound inhibition of CYP3A4 and only slow recovery after discontinuation. CLINICAL TRIAL REGISTRATION: EudraCT number: 2021-006643-39.
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Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2D6 , Citocromo P-450 CYP3A , Interacciones Farmacológicas , Inhibidores del Factor Xa , Voluntarios Sanos , Piridonas , Ritonavir , Humanos , Ritonavir/administración & dosificación , Ritonavir/farmacocinética , Ritonavir/farmacología , Masculino , Adulto , Inhibidores del Factor Xa/farmacocinética , Inhibidores del Factor Xa/administración & dosificación , Citocromo P-450 CYP3A/metabolismo , Piridonas/farmacocinética , Piridonas/administración & dosificación , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C19/genética , Administración Oral , Femenino , Rivaroxabán/farmacocinética , Rivaroxabán/administración & dosificación , Adulto Joven , Piridinas/farmacocinética , Piridinas/administración & dosificación , Piridinas/farmacología , Pirazoles/farmacocinética , Pirazoles/administración & dosificación , Pirazoles/farmacología , Tiazoles/farmacocinética , Tiazoles/administración & dosificación , Tiazoles/farmacología , Midazolam/farmacocinética , Midazolam/administración & dosificación , Omeprazol/farmacocinética , Omeprazol/administración & dosificación , Omeprazol/farmacologíaRESUMEN
The most important dose-limiting factor of the anthracycline idarubicin is the high risk of cardiotoxicity, in which the secondary alcohol metabolite idarubicinol plays an important role. It is not yet clear which enzymes are most important for the formation of idarubicinol and which inhibitors might be suitable to suppress this metabolic step and thus would be promising concomitant drugs to reduce idarubicin-associated cardiotoxicity. We, therefore, established and validated a mass spectrometry method for intracellular quantification of idarubicin and idarubicinol and investigated idarubicinol formation in different cell lines and its inhibition by known inhibitors of the aldo-keto reductases AKR1A1, AKR1B1, and AKR1C3 and the carbonyl reductases CBR1/3. The enzyme expression pattern differed among the cell lines with dominant expression of CBR1/3 in HEK293 and MCF-7 and very high expression of AKR1C3 in HepG2 cells. In HEK293 and MCF-7 cells, menadione was the most potent inhibitor (IC50 = 1.6 and 9.8 µM), while in HepG2 cells, ranirestat was most potent (IC50 = 0.4 µM), suggesting that ranirestat is not a selective AKR1B1 inhibitor, but also an AKR1C3 inhibitor. Over-expression of AKR1C3 verified the importance of AKR1C3 for idarubicinol formation and showed that ranirestat is also a potent inhibitor of this enzyme. Taken together, our study underlines the importance of AKR1C3 and CBR1 for the reduction of idarubicin and identifies potent inhibitors of metabolic formation of the cardiotoxic idarubicinol, which should now be tested in vivo to evaluate whether such combinations can increase the cardiac safety of idarubicin therapies while preserving its efficacy.
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Cardiotoxicidad , Daunorrubicina/análogos & derivados , Idarrubicina , Pirazinas , Compuestos de Espiro , Humanos , Idarrubicina/toxicidad , Idarrubicina/metabolismo , Aldo-Ceto Reductasas , Células HEK293 , Aldehído ReductasaRESUMEN
Tacrolimus is metabolized by cytochrome P450 3A (CYP3A) and is susceptible to interactions with the CYP3A and P-glycoprotein inducer St. John's Wort (SJW). CYP3A isozymes are predominantly expressed in the small intestine and liver. Prolonged-release tacrolimus (PR-Tac) is largely absorbed in distal intestinal segments and is less susceptible to CYP3A inhibition. The effect of induction by SJW is unknown. In this randomized, crossover trial, 18 healthy volunteers received single oral tacrolimus doses (immediate-release [IR]-Tac or PR-Tac, 5 mg each) alone and during induction by SJW. Concentrations were quantified using ultra-high performance liquid chromatography coupled with tandem mass spectrometry and non-compartmental pharmacokinetics were evaluated. SJW decreased IR-Tac exposure (area under the concentration-time curve) to 73% (95% confidence interval 60%-88%) and maximum concentration (Cmax ) to 61% (52%-73%), and PR-Tac exposure to 67% (55%-81%) and Cmax to 69% (58%-82%), with no statistical difference between the 2 formulations. The extent of interaction appeared to be less pronounced in volunteers with higher baseline CYP3A4 activity and in CYP3A5 expressors. In contrast to CYP3A inhibition, CYP3A induction by SJW showed a similar extent of interaction with both tacrolimus formulations. A higher metabolic baseline capacity appeared to attenuate the extent of induction by SJW.
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Hypericum , Tacrolimus , Humanos , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas , Hypericum/química , Hypericum/metabolismo , Extractos Vegetales , Tacrolimus/farmacocinética , Estudios CruzadosRESUMEN
Short bowel syndrome (SBS) following extensive intestinal resection is often characterized by impaired absorption of orally administered drugs, including tyrosine kinase inhibitors (TKI). We report the case of a patient with EGFR-mutated non-small cell lung carcinoma treated with 80 mg/day of the TKI osimertinib who achieved partial response of the tumour, but was subsequently subjected to a double-barrelled jejunostomy due to ileus. Due to the development of SBS after the bypass surgery, plasma concentrations of osimertinib were monitored using mass spectrometry. The therapeutic drug monitoring confirmed a malabsorption of osimertinib in the patient (108 ng/mL, which is below the 5th percentile of the expected plasma concentration) and was useful to guide adjustments of TKI dosing in order to achieve adequate blood levels (161 ng/mL after increase of the dose to 120 mg/day) in order to maintain tumour control.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Síndrome del Intestino Corto , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Síndrome del Intestino Corto/tratamiento farmacológico , Monitoreo de Drogas , Mutación , Receptores ErbB/genética , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
OBJECTIVE: To test feasibility and safety of administering sildenafil in neonates with neonatal encephalopathy (NE), developing brain injury despite therapeutic hypothermia (TH). STUDY DESIGN: We performed a randomized, double-blind, placebo-controlled phase Ib clinical trial between 2016 and 2019 in neonates with moderate or severe NE, displaying brain injury on day-2 magnetic resonance imaging (MRI) despite TH. Neonates were randomized (2:1) to 7-day sildenafil or placebo (2 mg/kg/dose enterally every 12 hours, 14 doses). Outcomes included feasibility and safety (primary outcomes), pharmacokinetics (secondary), and day-30 neuroimaging and 18-month neurodevelopment assessments (exploratory). RESULTS: Of the 24 enrolled neonates, 8 were randomized to sildenafil and 3 to placebo. A mild decrease in blood pressure was reported in 2 of the 8 neonates after initial dose, but not with subsequent doses. Sildenafil plasma steady-state concentration was rapidly reached, but decreased after TH discontinuation. Twelve percent of neonates (1/8) neonates died in the sildenafil group and 0% (0/3) in the placebo group. Among surviving neonates, partial recovery of injury, fewer cystic lesions, and less brain volume loss on day-30 magnetic resonance imaging were noted in 71% (5/7) of the sildenafil group and in 0% (0/3) of the placebo group. The rate of death or survival to 18 months with severe neurodevelopmental impairment was 57% (4/7) in the sildenafil group and 100% (3/3) in the placebo group. CONCLUSIONS: Sildenafil was safe and well-absorbed in neonates with NE treated with TH. Optimal dosing needs to be established. Evaluation of a larger number of neonates through subsequent phases II and III trials is required to establish efficacy. CLINICAL TRIAL REGISTRATION: ClinicalTrials.govNCT02812433.
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Asfixia Neonatal , Lesiones Encefálicas , Hipotermia Inducida , Hipoxia-Isquemia Encefálica , Enfermedades del Recién Nacido , Recién Nacido , Humanos , Citrato de Sildenafil/efectos adversos , Asfixia/complicaciones , Estudios de Factibilidad , Asfixia Neonatal/terapia , Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/tratamiento farmacológico , Enfermedades del Recién Nacido/terapia , Hipoxia-Isquemia Encefálica/terapia , Hipotermia Inducida/métodos , Método Doble CiegoRESUMEN
BACKGROUND AND OBJECTIVE: Although polypharmacy is a particular challenge in daily rheumatological practice, clinical research on the effects of hydroxychloroquine (HCQ), a commonly used drug for patients with rheumatic diseases, is sparse on cytochrome P450 (CYP)-mediated metabolism. We have shown that pre-treatment with pantoprazole does not alter HCQ absorption in healthy volunteers. In this paper, we report the effects of a single 400 mg dose of HCQ on specific CYP3A and CYP2D6 substrates in healthy volunteers. METHODS: In the trial, participants were randomized into two groups (HCQ plus a 9-day course of pantoprazole, or HCQ only). As a secondary endpoint, the effects of a single oral dose of HCQ on the exposure of the oral microdosed CYP3A probe drug midazolam (30 µg) and the oral microdosed CYP2D6 probe drug yohimbine (50 µg) were studied in 23 healthy volunteers (EudraCT no. 2020-001470-30, registered 31 March 2020). RESULTS: The exposure of the probe drugs after intake of HCQ compared with baseline values was quantified by the partial area under the plasma concentration-time curve 0-6 h after administration (AUC0-6 h) for yohimbine and the partial AUC2-4 h for midazolam. Under HCQ, yohimbine AUC0-6 h was unchanged, independent of CYP2D6 genotypes and pantoprazole exposure. Midazolam AUC2-4 h was 25% higher on the day of HCQ administration than at baseline (p = 0.0007). This significant increase was driven by the pantoprazole subgroup, which showed a 46% elevation of midazolam AUC2-4 h as compared with baseline (p < 0.0001). The ratio of midazolam to 1-OH-midazolam partial AUC2-4 h significantly increased from 3.03 ± 1.59 (baseline) to 3.60 ± 1.56 (HCQ) in the pantoprazole group (p = 0.0026). CONCLUSION: In conclusion, we observed an increased midazolam exposure most likely related to pantoprazole.
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Citocromo P-450 CYP3A , Hidroxicloroquina , Humanos , Área Bajo la Curva , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Voluntarios Sanos , Hidroxicloroquina/farmacología , Hidroxicloroquina/uso terapéutico , Midazolam , Pantoprazol/farmacología , Preparaciones Farmacéuticas , YohimbinaRESUMEN
The calcium-dependent serine endoprotease PACE4 is evaluated as a therapeutic target for prostate cancer. The peptide Ac-[d-Leu]LLLRVK-amba inhibits PACE4 with high affinity and has shown efficacy in preclinical mice xenograft models of prostate cancer. To support in vivo examinations of the potential therapeutic peptide Ac-[d-Leu]LLLRVK-amba, we established a highly sensitive assay for its quantification in mouse whole blood microsamples based on UPLC-MS/MS determination. Ac-[d-Leu]LLLRVK-amba was very labile during sample processing, which was particularly pronounced in plasma. High resolution mass spectrometric investigations of the metabolism/degradation in plasma revealed that no peptide bond hydrolysis generated products were formed, leaving the cause of the observed consumption of the peptide elusive. As a consequence, whole-blood quantification was developed relying on the immediate snap-freezing of blood samples after collection and immediate sample processing after serial thawing to ensure accurate and reliable quantification. The assay was validated according to the applicable recommendations of the FDA and EMA in a range of 10-10,000 ng/mL and applied to determine the pharmacokinetics of Ac-[d-Leu]LLLRVK-amba after intravenous and intraperitoneal administration to mice. Individual pharmacokinetic profiles were assessed using four microsamplings per animal. Intraperitoneal absorption was found to be efficient, demonstrating that this well-manageable route of administration is feasible for preclinical efficacy experiments with Ac-[d-Leu]LLLRVK-amba.
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PURPOSE: Early antiviral treatment with nirmatrelvir/ritonavir is recommended for SARS-CoV-2-infected patients at high risk for severe courses. Such patients are usually chronically ill and susceptible to adverse drug interactions caused by ritonavir. We investigated the interactions of short-term low-dose ritonavir therapy with atorvastatin and rosuvastatin, two statins commonly used in this population. METHOD: We assessed exposure changes (area under the concentration-time curve (AUC∞) and maximum concentration (Cmax)) of a single dose of 10 mg atorvastatin and 10 mg rosuvastatin before and on the fifth day of ritonavir treatment (2 × 100 mg/day) in healthy volunteers and developed a semi-mechanistic pharmacokinetic model to estimate dose adjustment of atorvastatin during ritonavir treatment. RESULTS: By the fifth day of ritonavir treatment, the AUC∞ of atorvastatin increased 4.76-fold and Cmax 3.78-fold, and concurrently, the concentration of atorvastatin metabolites decreased to values below the lower limit of quantification. Pharmacokinetic modelling indicated that a stepwise reduction in atorvastatin dose during ritonavir treatment with a stepwise increase up to 4 days after ritonavir discontinuation can keep atorvastatin exposure within safe and effective margins. Rosuvastatin pharmacokinetics were only mildly modified; ritonavir significantly increased the Cmax 1.94-fold, while AUC∞ was unchanged. CONCLUSION: Atorvastatin doses should likely be adjusted during nirmatrelvir/ritonavir treatment. For patients on a 20-mg dose, we recommend half of the original dose. In patients taking 40 mg or more, a quarter of the dose should be taken until 2 days after discontinuation of nirmatrelvir/ritonavir. Patients receiving rosuvastatin do not need to change their treatment regimen. TRIAL REGISTRATION: EudraCT number: 2021-006634-39. DRKS00027838.
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Expression of CYP3A5 protein is a basal and acquired resistance mechanism of pancreatic ductal adenocarcinoma cells conferring protection against the CYP3A and CYP2C8 substrate paclitaxel through metabolic degradation. Inhibition of CYP3A isozymes restores the cells sensitivity to paclitaxel. The combination of gemcitabine and nab-paclitaxel is an established regimen for the treatment of metastasized or locally advanced inoperable pancreatic cancer. Cobicistat is a CYP3A inhibitor developed for the pharmacoenhancement of protease inhibitors. The addition of cobicistat to gemcitabine and nab-paclitaxel may increase the antitumor effect. We will conduct a phase I dose escalation trial with a classical 3 + 3 design to investigate the safety, tolerability, and pharmacokinetics (PKs) of gemcitabine, nab-paclitaxel, and cobicistat. Although the doses of gemcitabine (1000 mg/m2 ) and cobicistat (150 mg) are fixed, three dose levels of nab-paclitaxel (75, 100, and 125 mg/m2 ) will be explored to account for a potential PK drug interaction. After the dose escalation phase, we will set the recommended dose for expansion (RDE) and treat up to nine patients in an expansion part of the trial. The trial is registered under the following identifiers EudraCT-Nr. 2019-001439-29, drks.de: DRKS00029409, and ct.gov: NCT05494866. Overcoming resistance to paclitaxel by CYP3A5 inhibition may lead to an increased efficacy of the gemcitabine and nab-paclitaxel regimen. Safety, efficacy, PK, and RDE data need to be acquired before investigating this combination in a large-scale clinical study.
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Carcinoma Ductal Pancreático , Citostáticos , Neoplasias Pancreáticas , Humanos , Gemcitabina , Citocromo P-450 CYP3A , Inhibidores del Citocromo P-450 CYP3A , Citostáticos/uso terapéutico , Desoxicitidina/efectos adversos , Cobicistat , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Paclitaxel/efectos adversos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Ensayos Clínicos Fase I como AsuntoRESUMEN
Neoadjuvant chemotherapy can improve the survival of individuals with borderline and unresectable pancreatic ductal adenocarcinoma; however, heterogeneous responses to chemotherapy remain a significant clinical challenge. Here, we performed RNA sequencing (n = 97) and multiplexed immunofluorescence (n = 122) on chemo-naive and postchemotherapy (post-CTX) resected patient samples (chemoradiotherapy excluded) to define the impact of neoadjuvant chemotherapy. Transcriptome analysis combined with high-resolution mapping of whole-tissue sections identified GATA6 (classical), KRT17 (basal-like) and cytochrome P450 3A (CYP3A) coexpressing cells that were preferentially enriched in post-CTX resected samples. The persistence of GATA6hi and KRT17hi cells post-CTX was significantly associated with poor survival after mFOLFIRINOX (mFFX), but not gemcitabine (GEM), treatment. Analysis of organoid models derived from chemo-naive and post-CTX samples demonstrated that CYP3A expression is a predictor of chemotherapy response and that CYP3A-expressing drug detoxification pathways can metabolize the prodrug irinotecan, a constituent of mFFX. These findings identify CYP3A-expressing drug-tolerant cell phenotypes in residual disease that may ultimately inform adjuvant treatment selection.
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Adenocarcinoma , Terapia Neoadyuvante , Humanos , Citocromo P-450 CYP3A , Adyuvantes Inmunológicos , Queratina-17 , FenotipoRESUMEN
Quantitative monitoring of biologically active methylations of guanines in samples exposed to temozolomide (TMZ) would be useful in glioblastoma research for preclinical TMZ experiments, for clinical pharmacology questions regarding appropriate exposure, and ultimately for precision oncology. The known biologically active alkylation of DNA induced by TMZ takes place on O6 position of guanines. However, when developing mass spectrometric (MS) assays, the possible signal overlap of O6-methyl-2'-deoxyguanosine (O6-m2dGO) with other methylated 2'-deoxyguanosine species in DNA and methylated guanosines in RNA must be considered. Liquid chromatography-tandem MS (LC-MS/MS) offers the analytical requirements for such assays in terms of specificity and sensitivity, especially when multiple reaction monitoring (MRM) is available. In preclinical research, cancer cell lines are still the gold standard model for in vitro drug screening. Here, we present the development of ultra-performance LC-MRM-MS assays for the quantification of O6-m2dGO in a TMZ-treated glioblastoma cell line. Furthermore, we propose adapted parameters for method validation relevant to the quantification of drug-induced DNA modifications.
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BACKGROUND AND OBJECTIVE: Voriconazole is an important broad-spectrum anti-fungal drug with nonlinear pharmacokinetics. The aim of this single centre fixed-sequence open-label drug-drug interaction trial in healthy participants (N = 17) was to determine whether microdosed probe drugs for CYP3A and CYP2C19 reliably predict voriconazole clearance (CLVRZ). METHODS: At baseline, a single oral microdose of the paradigm substrates midazolam (CYP3A) and omeprazole (CYP2C19) were given to estimate their clearances (CL). Thereafter, a single oral dose of voriconazole was administered (50, 100, 200 or 400 mg), followed by the microdosed probe drugs. RESULTS: The clearances of midazolam (CLMDZ 790-2790 mL/min at baseline; 248-1316 mL/min during voriconazole) and omeprazole (CLOMZ 66.4-2710 mL/min at baseline; 30.1-1420 mL/min during voriconazole) were highly variable. CLMDZ [geometric mean ratio (GMR) 0.586 at 50 mg voriconazole decreasing to GMR 0.196 at 400 mg voriconazole] and CLOMZ (GMR 0.590 at 50 mg decreasing to GMR 0.166 at 400 mg) were reduced with higher voriconazole doses. CLMDZ was linearly correlated with CLVRZ (slope 1.458; adjusted R2 0.528) as was CLOMZ (slope 0.807; adjusted R2 0.898). Multiple linear regression resulted in an adjusted R2 of 0.997 for the relationship CLVRZ ~ log CLOMZ + log CLMDZ using data during voriconazole treatment and an adjusted R2 of 0.997 for the relationship CLVRZ ~ log CLOMZ + log CLMDZ + voriconazole dose, using baseline data for CLMDZ and CLOMZ. CONCLUSION: Microdosed midazolam and omeprazole accurately described and predicted total CLVRZ TRIAL REGISTRATION: EudraCT No: 2020-001017-20, registered on March 5th, 2020. DRKS: DRKS00022547, registered on August 6th, 2020.
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Citocromo P-450 CYP3A , Midazolam , Humanos , Adulto , Voriconazol/farmacocinética , Midazolam/farmacocinética , Citocromo P-450 CYP2C19 , Omeprazol , Interacciones FarmacológicasRESUMEN
The development of desorption/ionization (DI) mass spectrometric (MS) assays for drug quantification in tissue sections and their validation according to regulatory guidelines would enable their universalization for applications in (clinical) pharmacology. Recently, new enhancements in desorption electrospray ionization (DESI) have highlighted the reliability of this ion source for the development of targeted quantification methods that meet requirements for method validation. However, it is necessary to consider subtle parameters leading to the success of such method developments, such as the morphology of desorption spots, the analytical time, and sample surface, to cite but a few. Here, we provide additional experimental data highlighting an additional important parameter, based on the unique advantage of DESI-MS on continuous extraction during analysis. We demonstrate that considering desorption kinetics during DESI analyses would largely help (i) reducing analytical time during profiling analyses, (ii) verifying solvent-based drug extraction using the selected sample preparation method for profiling and imaging modes, and (iii) predicting the feasibility of imaging assays using samples in a given expected concentration range of the targeted drug. These observations will likely serve as precious guidance for the development of validated DESI-profiling and imaging methods in the future.
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Diagnóstico por Imagen , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Ionización de Electrospray/métodos , Reproducibilidad de los Resultados , CinéticaRESUMEN
Introduction: Bulevirtide is a first-in-class antiviral drug to treat chronic hepatitis B/D. We investigated the drug-drug interaction potential and pharmacokinetics of high-dose subcutaneous bulevirtide (5 mg twice daily) with organic anion transporting polypeptide 1B1 (OATP1B1) and cytochrome P450 (CYP) 3A4. Methods: This was a single-center, open-label, fixed-sequence drug-drug interaction trial in 19 healthy volunteers. Before and at bulevirtide steady state, participants ingested a single 40 mg dose of pravastatin. A midazolam microdose was applied to quantify CYP3A4 activity. Results: At bulevirtide steady state, pravastatin area under the concentration-time curve (AUC0-∞) increased 1.32-fold (90% CI 1.08-1.61). The 5 mg bulevirtide twice-daily treatment resulted in a mean AUC0-12 of 1210 h*ng/ml (95% CI 1040-1408) and remained essentially unchanged under the influence of pravastatin. CYP3A4 activity did not change to a clinically relevant extent. As expected, total bile acids increased substantially (35-fold) compared to baseline during bulevirtide treatment. All study medication was well tolerated. Discussion: The study demonstrated that high-dose bulevirtide inhibited OATP1B-mediated hepatic uptake of the marker substrate pravastatin but the extent is considered clinically not relevant. Changes in CYP3A4 activity were also not clinically relevant. In conclusion, this study suggests that OATP1B substrate drugs as well as CYP3A4 substrates may safely be used without dose adjustment in patients treated with bulevirtide. However, in patients using high statin doses and where concomitant factors potentially further increase statin exposure, caution may be required when using bulevirtide.
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In clinical pharmacology, drug quantification is mainly performed from the circulation for pharmacokinetic purposes. Finely monitoring the chemical effect of drugs at their chemical sites of action for pharmacodynamics would have a major impact in several contexts of personalized medicine. Monitoring appropriate drug exposure is particularly challenging for alkylating drugs such as temozolomide (TMZ) because there is no flow equilibrium that would allow reliable conclusions to be drawn about the alkylation of the target site from plasma concentrations. During the treatment of glioblastoma, it appears, therefore, promising to directly monitor the alkylating effect of TMZ rather than plasma exposure, ideally at the site of action. Mass spectrometry (MS) is a method of choice for the quantification of methylated guanines and, more specifically, of O6-methylguanines as a marker of TMZ exposure at the site of action. Depending on the chosen strategy to analyze modified purines and 2'-deoxynucleosides, the analysis of methylated guanines and 2'-deoxyguanosines is prone to important artefacts due to the overlap between masses of (i) guanines from DNA and RNA, and (ii) different methylated species of guanines. Therefore, the specific analysis of O6-methyl-2'deoxyguanosine, which is the product of the TMZ effect, is highly challenging. In this work, we report observations from matrix-assisted laser desorption/ionization (MALDI), and desorption electrospray ionization (DESI) MS analyses. These allow for the construction of a decision tree to initiate studies using desorption/ionization MS for the analysis of 2'-deoxyguanosine methylations induced by TMZ.
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BACKGROUND: Frailty makes older adults vulnerable to adverse health outcomes and can modify pharmacokinetics and drug exposure. OBJECTIVE: We aimed to explore the relationship between different frailty assessments and trough plasma concentrations of direct oral anticoagulants in older patients. METHODS: The frailty status of adults aged ≥ 70 years receiving regular direct oral anticoagulant medication was assessed by four different instruments: Fried physical phenotype, Rockwood frailty index, Short Physical Performance Battery, and FRAIL scale. The two performance measures "slow gait speed" and "weak grip strength" were used to build a separate score depending on the number of positive criteria (none, one, two). For each participant, a single steady-state direct oral anticoagulant trough plasma concentration was collected, dose-normalized, and its relationship to the various frailty assessments analyzed. RESULTS: Forty-two participants completed the study, with most using apixaban (n = 22). Dose-normalized apixaban trough concentrations were 2.48-fold higher in frail participants (Fried phenotype) than in robust participants (p = 0.009) and correlated positively with Fried physical phenotype (rs = 0.535, p = 0.010) and negatively with Short Physical Performance Battery (rs = - 0.434, p = 0.044). Compared with participants who met none of the criteria "slow gait speed" and "weak grip strength", apixaban trough concentrations were approximately 1.9-fold higher in participants who were positive for one (p = 0.018) or two (p = 0.013) of these measures. CONCLUSIONS: In this exploratory study, higher levels of frailty on performance-based frailty assessments were associated with higher apixaban exposure in older adults. CLINICAL TRIAL REGISTRATION: German Clinical Trials Register DRKS00016741; registered 20 February, 2019.