Effect of Clomethiazole Vs. Clorazepate on Hepatic Fat and Serum Transaminase Activities in Alcohol-Associated Liver Disease: Results from a Randomized, Controlled Phase II Clinical Trial.

AIMS: Alcohol-associated liver disease (ALD) is a global health problem caused, among other factors, by oxidative stress from the formation of reactive oxygen species (ROS). One important source of ROS is microsomal ethanol metabolism catalyzed by cytochrome P450 2E1 (CYP2E1), which is induced by chronic ethanol consumption. Inhibition of CYP2E1 by clomethiazole (CMZ) decreases oxidative stress in cell cultures and improves ALD in animal studies. Our study aimed to assess the benefits of a CYP2E1 inhibitor (clomethiazole) in detoxification of patients with ALD. METHODS: Open label, randomized controlled clinical trial to study whether CYP2E1 inhibition improves ALD in the patients with alcohol use disorders admitted for alcohol detoxification therapy (ADT). Patients had to have a serum aspartate aminotransferase (AST) activity exceeding twice the upper normal limit at time of admission and be non-cirrhotic defined by fibroscan value <12 kPa. Sixty patients were randomly assigned to ADT with either CMZ or clorazepate (CZP) for 7-10 days in a 1:1 ratio. The chlorzoxazone test of CYP2E1 activity was performed at enrolment and at 2 points during the study. RESULTS: ADT improved hepatic steatosis (controlled attenuation parameter) in both groups significantly. A trend towards a greater improvement in hepatic fat content during ADT (-21.5%) was observed in the CMZ group (252 ± 48 dB/m vs. 321 ± 38 dB/m; P < 0.0001) compared with the CZP group (-13.9%; 273 ± 38 dB/m vs. 317 ± 39 dB/m; P < 0.0001). As already reported, serum AST (P < 0.004) and alanine aminotransferase (ALT) activities (P < 0.0006) significantly decreased in CMZ patients as compared with patients on CZP by the end of hospitalization. A significant correlation was found between AST (P = 0.023), ALT (P = 0.009), GGT (P = 0.039) and CAP. CONCLUSION: This study demonstrates that CMZ improves clinical biomarkers for ALD in humans most likely due to its inhibitory effect on CYP2E1. Because of its addictive potential, CMZ can only be given for a short period of time and therefore other CYP2E1 inhibitors to treat ALD are needed.

Molecular, Viral and Clinical Features of Alcohol- and Non-Alcohol-Induced Liver Injury.

Hepatic cells are sensitive to internal and external signals. Ethanol is one of the oldest and most widely used drugs in the world. The focus on the mechanistic engine of the alcohol-induced injury has been in the liver, which is responsible for the pathways of alcohol metabolism. Ethanol undergoes a phase I type of reaction, mainly catalyzed by the cytoplasmic enzyme, alcohol dehydrogenase (ADH), and by the microsomal ethanol-oxidizing system (MEOS). Reactive oxygen species (ROS) generated by cytochrome (CYP) 2E1 activity and MEOS contribute to ethanol-induced toxicity. We aimed to: (1) Describe the cellular, pathophysiological and clinical effects of alcohol misuse on the liver; (2) Select the biomarkers and analytical methods utilized by the clinical laboratory to assess alcohol exposure; (3) Provide therapeutic ideas to prevent/reduce alcohol-induced liver injury; (4) Provide up-to-date knowledge regarding the Corona virus and its affect on the liver; (5) Link rare diseases with alcohol consumption. The current review contributes to risk identification of patients with alcoholic, as well as non-alcoholic, liver disease and metabolic syndrome. Additional prevalence of ethnic, genetic, and viral vulnerabilities are presented.

A nuclear factor kappa B reporter cell line used to evaluate ex vivo the net inflammatory effect of plasma samples from patients with rheumatoid arthritis, psoriasis, or COVID-19.

BACKGROUND: The overall clinical outcome of inflammatory conditions is the result of the balance between pro-inflammatory and anti-inflammatory mediators. Because nuclear factor kappa B (NF-ĸB) is at the bottom of many inflammatory conditions, methods to evaluate the net effect of inflammation modulators on this master regulator have been conceptualized for years. METHODS: Using an ex vivo NF-ĸB reporter cell line-based assay, plasma samples of patients with rheumatoid arthritis (n = 27), psoriasis (n = 15), or severe coronavirus disease-19 (COVID-19) (n = 21) were investigated for NF-ĸB activation compared to plasma samples from 9 healthy volunteers. RESULTS: When separated by C-reactive protein (CRP) threshold levels, samples of patients exhibiting increased CRP levels (≥5 mg/l) activated NF-ĸB more efficiently than samples from patients with levels below 5 mg/l (P = 0.0001) or healthy controls (P = 0.04). Overall, there was a moderate association of CRP levels with NF-ĸB activation (Spearman r = 0.66; p < 0.0001). Plasma from COVID-19 patients activated NF-ĸB more efficiently (mean 2.4-fold compared to untreated reporter cells) than samples from any other condition (healthy controls, 1.8-fold, P = 0.0025; rheumatoid arthritis, 1.7-fold, P < 0.0001; psoriasis, 1.7-fold, P < 0.0001). In contrast, effects of rheumatoid arthritis, psoriasis, or healthy volunteer samples did not differ. CONCLUSION: This study shows that a NF-ĸB reporter cell line can be used to evaluate the net inflammatory effect of clinical plasma samples. Patients with chronic but stable rheumatoid arthritis or psoriasis do not exhibit increased plasma levels of NF-ĸB-activating compounds as opposed to COVID-19 patients with high inflammatory burden.

Simultaneous phenotyping of CYP2E1 and CYP3A using oral chlorzoxazone and midazolam microdoses.

AIMS: Chlorzoxazone is the paradigm marker substrate for CYP2E1 phenotyping in vivo. Because at the commonly used milligram doses (250-750 mg) chlorzoxazone acts as an inhibitor of the CYP3A4/5 marker substrate midazolam, previous attempts failed to combine both drugs in a common phenotyping cocktail. Microdosing chlorzoxazone could circumvent this problem. METHOD: We enrolled 12 healthy volunteers in a trial investigating the dose-exposure relationship of single ascending chlorzoxazone oral doses over a 10,000-fold range (0.05-500 mg) and assessed the effect of 0.1 and 500 mg of chlorzoxazone on oral midazolam pharmacokinetics (0.003 mg). RESULTS: Chlorzoxazone area under the concentration-time curve was dose-linear in the dose range between 0.05 and 5 mg. A nonlinear increase occurred with doses ≥50 mg, probably due to saturated presystemic metabolic elimination. While midazolam area under the concentration-time curve increased 2-fold when coadministered with 500 mg of chlorzoxazone, there was no pharmacokinetic interaction between chlorzoxazone and midazolam microdoses. CONCLUSION: The chlorzoxazone microdose did not interact with the CYP3A marker substrate midazolam, enabling the simultaneous administration in a phenotyping cocktail. This microdose assay is now ready to be further validated and tested as a phenotyping procedure assessing the impact of induction and inhibition of CYP2E1 on chlorzoxazone microdose pharmacokinetics.

Autoinhibitory properties of the parent but not of the N-oxide metabolite contribute to infusion rate-dependent voriconazole pharmacokinetics.

AIMS: The pharmacokinetics of voriconazole show a nonlinear dose-exposure relationship caused by inhibition of its own CYP3A-dependent metabolism. Because the magnitude of autoinhibition also depends on voriconazole concentrations, infusion rate might modulate voriconazole exposure. The impact of four different infusion rates on voriconazole pharmacokinetics was investigated. METHODS: Twelve healthy participants received 100 mg voriconazole intravenous over 4 h, 400 mg over 6 h, 4 h, and 2 h in a crossover design. Oral midazolam (3 µg) was given at the end of infusion. Blood and urine samples were collected up to 48 h. Voriconazole and its N-oxide metabolite were quantified using high-performance liquid chromatography coupled to tandem mass spectrometry. Midazolam estimated metabolic clearance (eCLmet) was calculated using a limited sampling strategy. Voriconazole-N-oxide inhibition of cytochrome P450 (CYP) isoforms 2C19 and 3A4 were assessed with the P450-Glo luminescence assay. RESULTS: Area under the concentration-time curve for 400 mg intravenous voriconazole was 16% (90% confidence interval: 12-20%) lower when administered over 6 h compared to 2 h infusion. Dose-corrected area under the concentration-time curve for 100 mg over 4 h was 34% lower compared to 400 mg over 4 h. Midazolam eCLmet was 516 ml min-1 (420-640) following 100 mg 4 h-1 voriconazole, 152 ml min-1 (139-166) for 400 mg 6 h-1 , 192 ml min-1 (167-220) for 400 mg 4 h-1 , and 202 ml min-1 (189-217) for 400 mg 2 h-1 . Concentration giving 50% CYP inhibition of voriconazole N-oxide was 146 ± 23 µmol l-1 for CYP3A4, and 40.2 ± 4.2 µmol l-1 for CYP2C19. CONCLUSIONS: Voriconazole pharmacokinetics is modulated by infusion rate, an autoinhibitory contribution voriconazole metabolism by CYP3A and 2C19 and to a lesser extent its main N-oxide metabolite for CYP2C19. To avoid reduced exposure, the infusion rate should be 2 h.

First-in-human application of the novel hepatitis B and hepatitis D virus entry inhibitor myrcludex B.

BACKGROUND & AIMS: Myrcludex B is a first-in-class compound, which blocks entry of hepatitis B and D virus into hepatocytes in vitro and in animal models. Based on the required preclinical data we aimed to translate this compound into the first application in humans. METHODS: Single ascending doses of myrcludex B, a 47 amino acid peptide, were administered up to 20mg intravenously and 10mg subcutaneously in a prospective open first-in-human, phase I clinical trial to 36 healthy volunteers. Safety, tolerability and plasma concentrations of myrcludex B were assessed and a pharmacokinetic model was derived. RESULTS: Myrcludex B was well tolerated and no serious or relevant AEs representing off-target effects, and no immunogenic effects were observed up to the highest applied dose of 20mg (intravenously). Myrcludex B showed dose-dependent pharmacokinetics, best described by a 2-compartment target-mediated drug disposition model. Bioavailability of the subcutaneous application was large (85%). Interindividual variability was moderate. The pharmacokinetic model suggested that subcutaneous doses of 10mg and above reach a target saturation of over 80% for at least 15h. CONCLUSIONS: Myrcludex B showed excellent tolerability up to high doses. Pharmacologic properties followed a 2-compartment target-mediated drug disposition model. These findings are vital for planning of further multiple dose efficacy trials in patients. LAY SUMMARY: After showing antiviral activity in cell culture and animal models, myrcludex B, a new drug intended for the treatment of hepatitis B and D, has been administered the first time in humans. Healthy volunteers received the drug intravenously and subcutaneously up to high doses (20mg). The drug was well tolerated and the characteristics of the drug determining its way in the human body could be described. These results will allow testing myrcludex B in hepatitis B and D patients.

Treatment with rilpivirine does not alter plasma concentrations of the CYP3A substrates tadalafil and midazolam in humans.

OBJECTIVES: Antiretroviral combination therapy of patients infected with HIV has greatly increased their life expectancy. Hence, the treatment of HIV-related long-term complications and age-related comorbidities has become more important. Reported incidence rates of erectile dysfunction (ED) and pulmonary arterial hypertension (PAH) are increasing in HIV-positive patients, potentially requiring treatment with phosphodiesterase 5 inhibitors such as sildenafil or tadalafil. In vitro, the NNRTI rilpivirine is both a pregnane X receptor agonist and cytochrome P450 (CYP) 3A inhibitor. Clinical data concerning the potential effects of rilpivirine coadministration on the pharmacokinetics of the CYP3A substrate tadalafil are lacking. METHODS: We enrolled 20 healthy volunteers in an open-label, two-part, one-arm Phase I clinical trial to investigate acute and chronic effects of multiple doses of 25 mg of oral rilpivirine on single-dose and steady-state pharmacokinetics of multiple oral 20 mg doses of tadalafil. CYP3A activity was measured simultaneously with the oral midazolam microdose test. RESULTS: We did not observe a change of tadalafil single-dose and steady-state exposure or of CYP3A activity measured at initiation, during maintenance and upon discontinuation of rilpivirine treatment after single-dose and chronic administration of rilpivirine. CONCLUSIONS: Tadalafil can be combined with rilpivirine without dose adjustment or drug monitoring in HIV patients with ED or PAH. Rilpivirine at daily therapeutic doses of 25 mg does not induce or inhibit CYP3A-dependent drug metabolism.

St. John's wort treatment in women bears risks beyond pharmacokinetic drug interactions.

We analyzed adverse events in a clinical phase I trial to assess dose-dependent metabolic effects of St. John's wort co-administered with rifampicin in 12 healthy volunteers. Within 3-6 days after increasing the St. John's wort dose from 300 to 600 mg TID, five of six female participants developed ambient temperature-dependent allodynia and paresthesia in sun-exposed areas (back of the hands and perioral and nasal area). Aggravation of symptoms resulted in persistence of paresthesia and phototoxic erythrodermia. None of the male participants showed any of these effects. Gender, duration of treatment, dose, and solar exposure seem to be extrinsic and host factors facilitating St. John's wort-induced neuropathy. The risk to develop this adverse effect is almost exclusively present in women.

Estrogen Receptor Signaling and the PI3K/Akt Pathway Are Involved in Betulinic Acid-Induced eNOS Activation.

Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid with anti-inflammatory, antiviral and anti-cancer properties. Beneficial cardiovascular effects such as increased nitric oxide (NO) production through enhancement of endothelial NO synthase (eNOS) activity and upregulation of eNOS expression have been demonstrated for this compound. In the present study, immortalized human EA.hy 926 endothelial cells were incubated for up to 1 h with 1-100 µM BA and with the phosphatidylinositol-3-kinase (PI3K) inhibitors LY294002 and wortmannin, or the estrogen receptor (ER) antagonist ICI 182,780. Phosphorylation status of eNOS and total eNOS protein were analyzed by Western blotting using a serine 1177 phosphosite-specific antibody. Bioactive NO production was assessed by determination of cGMP content in rat lung fibroblasts (RFL-6) reporter cells. Short-term incubation of EA.hy 926 cells with BA resulted in eNOS phosphorylation at the serine 1177 residue in a concentration- and time-dependent manner with a half-maximal effective concentration of 0.57 µM. This was associated with an enhanced production of NO. BA-induced eNOS phosphorylation and NO production was completely blocked by pretreatment with ICI 182,780, and was attenuated by pretreatment with the PI3K inhibitors wortmannin and LY294002. These results indicate that fast non-genomic effects of ER with downstream signaling through the PI3K/Akt pathway and consecutive eNOS phosphorylation at serine 1177 are involved in BA-induced eNOS activation.

Midazolam microdose to determine systemic and pre-systemic metabolic CYP3A activity in humans.

AIM: We aimed to establish a method to assess systemic and pre-systemic cytochrome P450 (CYP) 3A activity using ineffective microgram doses of midazolam. METHODS: In an open, one sequence, crossover study, 16 healthy participants received intravenous and oral midazolam at microgram (0.001 mg intravenous and 0.003 mg oral) and regular milligram (1 mg intravenous and 3 mg oral) doses to assess the linearity of plasma and urine pharmacokinetics. RESULTS: Dose-normalized AUC and Cmax were 37.1 ng ml(-1 ) h [95% CI 35.5, 40.6] and 39.1 ng ml(-1) [95% CI 30.4, 50.2] for the microdose and 39.0 ng ml(-1 ) h [95% CI 36.1, 42.1] and 37.1 ng ml(-1) [95% CI 26.9, 51.3] for the milligram dose. CLmet was 253 ml min(-1) [95% CI 201, 318] vs. 278 ml min(-1) [95% CI 248, 311] for intravenous doses and 1880 ml min(-1) [95% CI 1590, 2230] vs. 2050 ml min(-1) [95% CI 1720, 2450] for oral doses. Oral bioavailability of a midazolam microdose was 23.4% [95% CI 20.0, 27.3] vs. 20.9% [95% CI 17.1, 25.5] after the regular dose. Hepatic and gut extraction ratios for microgram doses were 0.44 [95% CI 0.39, 0.49] and 0.53 [95% CI 0.45, 0.63] and compared well with those for milligram doses (0.43 [95% CI 0.37, 0.49] and 0.61 [95% CI 0.53, 0.70]). CONCLUSION: The pharmacokinetics of an intravenous midazolam microdose is linear to the applied regular doses and can be used to assess safely systemic CYP3A activity and, in combination with oral microdoses, pre-systemic CYP3A activity.

Systemic exposure of topical erythromycin in comparison to oral administration and the effect on cytochrome P450 3A4 activity.

AIMS: Erythromycin is a macrolide antibiotic, which is frequently used as a topical formulation for the treatment of acne. It is also known as an inhibitor of the cytochrome P450 (CYP) isoenzyme 3A4. In this study, the systemic availability of topical erythromycin, hence the influence on the activity of CYP3A, is evaluated in comparison to orally administered erythromycin. METHODS: Sixteen healthy volunteers received consecutively topical (two applications of 800 mg) and oral erythromycin (two dose groups, 250 and 1000 mg, with n = 8) to assess erythromycin pharmacokinetics. A microdose of midazolam (3 µg orally) was used to determine the effect on CYP3A activity. RESULTS: After topical administration, erythromycin was detected in the plasma of every participant without causing a statistically significant alteration of CYP3A activity. After oral administration, the dose-normalized erythromycin exposure (AUC∞ ) was 1335 h ng ml(-1) after 250 mg and 3-fold higher after the 1000 mg dose (4051 h ng ml(-1); P < 0.01), suggesting nonlinear pharmacokinetics of erythromycin. Both oral doses inhibited CYP3A activity; midazolam clearance was decreased to 61% (250 mg) and 21% (1000 mg). The relationship between erythromycin exposure and CYP3A activity (Hill equation) revealed a 50% reduction of CYP3A activity by an erythromycin AUC∞ of 2106 h ng ml(-1). CONCLUSIONS: Topical erythromycin did not cause clinically relevant CYP3A alterations, although low systemic availability of erythromycin was observed. This supports the assumption that treatment with topical erythromycin is not critical in terms of CYP3A inhibition. Furthermore, substantial nonlinearity of erythromycin pharmacokinetics after two different oral doses was observed, possibly due to autoinhibition.

Dose-dependent induction of CYP3A activity by St. John's wort alone and in combination with rifampin.

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.

Protocol of the IntenSify-Trial: An open-label phase I trial of the CYP3A inhibitor cobicistat and the cytostatics gemcitabine and nab-paclitaxel in patients with advanced stage or metastatic pancreatic ductal adenocarcinoma to evaluate the combination's pharmacokinetics, safety, and efficacy.

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.

Persister cell phenotypes contribute to poor patient outcomes after neoadjuvant chemotherapy in PDAC.

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.

Double-blind, placebo-controlled first in human study to investigate an oral vaccine aimed to elicit an immune reaction against the VEGF-Receptor 2 in patients with stage IV and locally advanced pancreatic cancer.

BACKGROUND: The investigational oral DNA vaccine VXM01 targets the vascular endothelial growth factor receptor 2 (VEGFR-2) and uses Salmonella typhi Ty21a as a vector. The immune reaction elicited by VXM01 is expected to disrupt the tumor neovasculature and, consequently, inhibit tumor growth. VXM01 potentially combines the advantages of anti-angiogenic therapy and active immunotherapy. METHODS/DESIGN: This phase I trial examines the safety, tolerability, and immunological and clinical responses to VXM01. The randomized, placebo-controlled, double blind dose-escalation study includes up to 45 patients with locally advanced and stage IV pancreatic cancer. The patients will receive four doses of VXM01 or placebo in addition to gemcitabine as standard of care. Doses from 106 cfu up to 1010 cfu of VXM01 will be evaluated in the study. An independent data safety monitoring board (DSMB) will be involved in the dose-escalation decisions. In addition to safety as primary endpoint, the VXM01-specific immune reaction, as well as clinical response parameters will be evaluated. DISCUSSION: The results of this study shall provide the first data regarding the safety and immunogenicity of the oral anti-VEGFR-2 vaccine VXM01 in cancer patients. They will also define the recommended dose for phase II and provide the basis for further clinical evaluation, which may also include additional cancer indications. TRIAL REGISTRATION: EudraCT No.: 2011-000222-29, NCT01486329, ISRCTN68809279.

Quantification of femtomolar concentrations of the CYP3A substrate midazolam and its main metabolite 1'-hydroxymidazolam in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry.

The benzodiazepine midazolam is a probe drug used to phenotype cytochrome P450 3A activity. In this situation, effective sedative concentrations are neither needed nor desired, and in fact the use of very low doses is advantageous. We therefore developed and validated an assay for the femtomolar quantification of midazolam and 1'-hydroxymidazolam in human plasma. Plasma (0.25 mL) and 96-well-based solid-phase extraction were used for sample preparation. Extraction recoveries ranged between 75 and 92% for both analytes. Extracts were chromatographed within 2 min on a Waters BEH C18 1.7 µm UPLC® column with a fast gradient consisting of formic acid, ammonia, and acetonitrile. Midazolam and 1'-hydroxymidazolam were quantified using deuterium- and (13)C-labeled internal standards and positive electrospray tandem mass spectrometry in the multiple reaction monitoring mode, which yielded lower limits of quantification of 50 fg/mL (154 fmol/L) and 250 fg/mL (733 fmol/L) and a corresponding precision of <20%. The calibrated concentration ranges were linear for midazolam (0.05-250 pg/mL) and 1'-hydroxymidazolam (0.25-125 pg/mL), with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <14% and <12%. No ion suppression was detectable, and plasma matrix effects were minimized to <15% (<25%) for midazolam (1'-hydroxymidazolam). The assay was successfully applied to assess the kinetics of midazolam in two human volunteers after the administration of single oral microgram doses (1-100 µg). This ultrasensitive assay allowed us to quantify the kinetics of midazolam and 1'-hydroxymidazolam for at least 10 h, even after the administration of only 1 µg of midazolam.

Quantification of the Time Course of CYP3A Inhibition, Activation, and Induction Using a Population Pharmacokinetic Model of Microdosed Midazolam Continuous Infusion.

BACKGROUND: Cytochrome P450 (CYP) 3A contributes to the metabolism of many approved drugs. CYP3A perpetrator drugs can profoundly alter the exposure of CYP3A substrates. However, effects of such drug-drug interactions are usually reported as maximum effects rather than studied as time-dependent processes. Identification of the time course of CYP3A modulation can provide insight into when significant changes to CYP3A activity occurs, help better design drug-drug interaction studies, and manage drug-drug interactions in clinical practice. OBJECTIVE: We aimed to quantify the time course and extent of the in vivo modulation of different CYP3A perpetrator drugs on hepatic CYP3A activity and distinguish different modulatory mechanisms by their time of onset, using pharmacologically inactive intravenous microgram doses of the CYP3A-specific substrate midazolam, as a marker of CYP3A activity. METHODS: Twenty-four healthy individuals received an intravenous midazolam bolus followed by a continuous infusion for 10 or 36 h. Individuals were randomized into four arms: within each arm, two individuals served as a placebo control and, 2 h after start of the midazolam infusion, four individuals received the CYP3A perpetrator drug: voriconazole (inhibitor, orally or intravenously), rifampicin (inducer, orally), or efavirenz (activator, orally). After midazolam bolus administration, blood samples were taken every hour (rifampicin arm) or every 15 min (remaining study arms) until the end of midazolam infusion. A total of 1858 concentrations were equally divided between midazolam and its metabolite, 1'-hydroxymidazolam. A nonlinear mixed-effects population pharmacokinetic model of both compounds was developed using NONMEM®. CYP3A activity modulation was quantified over time, as the relative change of midazolam clearance encountered by the perpetrator drug, compared to the corresponding clearance value in the placebo arm. RESULTS: Time course of CYP3A modulation and magnitude of maximum effect were identified for each perpetrator drug. While efavirenz CYP3A activation was relatively fast and short, reaching a maximum after approximately 2-3 h, the induction effect of rifampicin could only be observed after 22 h, with a maximum after approximately 28-30 h followed by a steep drop to almost baseline within 1-2 h. In contrast, the inhibitory impact of both oral and intravenous voriconazole was prolonged with a steady inhibition of CYP3A activity followed by a gradual increase in the inhibitory effect until the end of sampling at 8 h. Relative maximum clearance changes were +59.1%, +46.7%, -70.6%, and -61.1% for efavirenz, rifampicin, oral voriconazole, and intravenous voriconazole, respectively. CONCLUSIONS: We could distinguish between different mechanisms of CYP3A modulation by the time of onset. Identification of the time at which clearance significantly changes, per perpetrator drug, can guide the design of an optimal sampling schedule for future drug-drug interaction studies. The impact of a short-term combination of different perpetrator drugs on the paradigm CYP3A substrate midazolam was characterized and can define combination intervals in which no relevant interaction is to be expected. CLINICAL TRIAL REGISTRATION: The trial was registered at the European Union Drug Regulating Authorities for Clinical Trials (EudraCT-No. 2013-004869-14).

First-in-human, randomized, double-blind, placebo-controlled, dose escalation trial of the anti-herpes simplex virus monoclonal antibody HDIT101 in healthy volunteers.

HDIT101 is a first-in-class humanized monoclonal antibody recognizing a conserved epitope in glycoprotein B, a target present on the surface of herpes simplex virus 1 (HSV-1) and HSV-2 particles as well as on virus-infected cells. This was a first-in-human, single-center, double-blind, placebo-controlled trial in 24 healthy volunteers, randomized 3:1 (placebo:active) in each of the six dose levels with escalating doses up to 12,150 mg HDIT101. HDIT101 was administered intravenously, to study safety, pharmacokinetics (PKs), and immunogenicity. HDIT101 was well-tolerated in all recipients and no serious or severe adverse events, no infusion-related reactions, and no events suggestive of dose limiting off-target toxicity occurred. The mean serum exposure (area under the curve from zero to infinity [AUC0-∞ ]) of HDIT101 showed a linear increase from 4340 h*µg/ml at a dose of 50 mg to 1,122,247 h*µg/ml at a dose of 12,150 mg. No immunogenic effects following HDIT101 exposure were observed at any of the applied doses. HDIT101 demonstrated the expected PK properties of a monoclonal antibody was well-tolerated, and could be safely administered even at excessively high doses that may be required for treatment of patients with septical HSV spread.

Pharmacoenhancement of Low Crizotinib Plasma Concentrations in Patients with Anaplastic Lymphoma Kinase-Positive Non-Small Cell Lung Cancer using the CYP3A Inhibitor Cobicistat.

The inhibitor of anaplastic lymphoma kinase (ALK) crizotinib significantly increases survival in patients with ALK-positive non-small cell lung cancer (NSCLC). When evaluating crizotinib pharmacokinetics (PKs) in patients taking the standard flat oral dose of 250 mg b.i.d., interindividual PK variability is substantial and patient survival is lower in the quartile with the lowest steady-state trough plasma concentrations (Cmin,ss ), suggesting that concentrations should be monitored and doses individualized. We investigated whether the CYP3A inhibitor cobicistat increases Cmin,ss of the CYP3A substrate crizotinib in patients with low exposure. Patients with ALK-positive NSCLC of our outpatient clinic treated with crizotinib were enrolled in a phase I trial (EudraCT 2016-002187-14, DRKS00012360) if crizotinib Cmin,ss was below 310 ng/mL and treated with cobicistat for 14 days. Crizotinib plasma concentration profiles were established before and after a 14-day co-administration of cobicistat to construct the area under the plasma concentration-time curve in the dosing interval from zero to 12 hours (AUC0-12 ). Patients were also monitored for adverse events by physical examination, laboratory tests, and 12-lead echocardiogram. Enrolment was prematurely stopped because of the approval of alectinib, a next-generation ALK-inhibitor with superior efficacy. In the only patient enrolled, cobicistat increased Cmin,ss from 158 ng/mL (before cobicistat) to 308 ng/mL (day 8) and 417 ng/mL (day 14 on cobicistat), concurrently the AUC0-12 increased by 78% from 2,210 ng/mL*h to 3,925 ng/mL*h. Neither safety signals nor serious adverse events occurred. Pharmacoenhancement with cobicistat as an alternative for dose individualisation for patients with NSCLC with low crizotinib exposure appears to be safe and is cost-effective and feasible.