Comparison between cytochrome P450 (CYP) content and relative activity approaches to scaling from cDNA-expressed CYPs to human liver microsomes: ratios of accessory proteins as sources of discrepancies between approaches. Drug Metab

This paper is available online at http://www.dmd.org ABSTRACT: Relative activity factors (RAFs) and immunoquantified levels of cytochrome P450 (CYP) isoforms both have been proposed as scaling factors for the prediction of hepatic drug metabolism from studies using cDNA-expressed CYPs. However, a systematic comparison of the two approaches, including possible mechanisms underlying differences, is not available. In this study, RAFs determined for CYPs 1A2, 2B6, 2C19, 2D6, and 3A4 in 12 human livers using lymphoblast-expressed enzymes were compared to immunoquantified protein levels. 2C19, 2D6, and 3A4 RAFs were similar to immunoquantified enzyme levels. In contrast, 1A2 RAFs were 5-to 20-fold higher than CYP1A2 content, and the RAF:content ratio was positively correlated with the molar ratio of NADPH:CYP oxidoreductase (OR) to CYP1A2. The OR:CYP1A2 ratio in lymphoblast microsomes was 92-fold lower than in human liver microsomes. Reconstitution experiments demonstrated a 10-to 20-fold lower activity at OR:CYP1A2 ratios similar to those in lymphoblasts, compared with those in human livers. CYP2B6-containing lymphoblast microsomes had 29-and 13-fold lower OR:CYP and cytochrome b 5 :CYP ratios, respectively, than did liver microsomes and yielded RAFs that were 6-fold higher than CYP2B6 content. Use of metabolic rates from cDNA-expressed CYPs containing nonphysiologic concentrations of electron-transfer proteins (relative to human liver microsomes) in conjunction with hepatic CYP contents may lead to incorrect predictions of liver microsomal rates and relative contributions of individual isoforms. Scaling factors used in bridging the gap between expression systems and liver microsomes should not only incorporate relative hepatic abundance of individual CYPs but also account for differences in activity per unit enzyme in the two systems. The cloning and heterologous expression of the drug-metabolizing human CYPs 1 has resulted in the commercial availability of cDNAexpressed CYPs for use as reagents for in vitro quantitative phenotyping, and bridging the gap between cDNA-expressed cytochromes and human liver microsomes has been the subject of several recent reviews The turnover number of CYPs in human liver microsomes is affected by several factors that are biochemically distinct from the CYP enzyme itself. Examples include the accessory electron-transfer proteins NADPH cytochrome P450-oxidoreductase (OR) and cytochrome b 5 , membrane lipid composition, and ionic strength of the in vitro incubation matrix The relative contribution of each isoform can then be determined as follows: Enzyme kinetic studies using cDNA-expressed human CYPs are being increasingly used to define the functions v i (s) for whatever individual CYPs biotransform a drug. Such analyses indicate the relative affinities and capacities of the CYPs contributing to a biotransformation. However, the relative contribution of each enzyme cannot be estimated unless the results incorporate the scaling factors (A i ). Although immunoquantified levels of CYP isoforms in human liver microsomes RAFs are determined for specific CYPs by comparing the rate of an isoform-specific index reaction at saturating substrate concentrations in human liver microsomes (V max for liver microsomes) to the rate of the same reaction catalyzed by the specific cDNA-expressed CYP under identical conditions (V max for cDNA-expressed enzyme): Mean V max for isoform-specific reaction in human liver microsomes V max for isoform-specific reaction by cDNA expressed isoform When the numerator is expressed in picomoles metabolite per milligram of protein per minute and the denominator as picomoles of metabolite per picomole of CYP per minute, the units of RAF are picomoles of CYP per milligram of protein, which can be compared with the immunoquantified CYP contents. RAFs have been used as estimates of A i in reaction phenotyping Materials and Methods Human Liver Microsomes and Heterologously Expressed CYP Isoforms. Liver samples, obtained from the International Institute for the Advancement of Medicine (Exton, PA) or the Liver Tissue Procurement and Distribution Service (University of Minnesota), were from 12 different transplant donors (L1-L12) with no known liver disease. The donor population (median age 27 years, range 3-50 years; 5 females and 7 males) was balanced with respect to age, sex, race, smoking habits, and alcohol consumption. The tissue was partitioned and kept at Ϫ80°C until the time of microsome preparation as described previously Microsomes from cDNA-transfected human lymphoblastoid cells expressing CYP 1A2, 2B6, 2C19, 2D6, or 3A4 (Crespi, 1995) were purchased from Gentest Corporation (Woburn, MA), aliquoted and stored at Ϫ80°C, and thawed on ice before use. Lymphoblast-expressed CYPs 3A4 and 2D6 used in the study were coexpressed with OR, whereas the activities of lymphoblast- expressed CYPs 1A2, 2B6, and 2C19 were supported by endogenous levels of reductase native to the host cell line. Microsomal protein concentrations and CYP content were provided by the manufacturer. Antibodies and Quantitative Western Blotting. Concentrations of CYP1A2, 2B6, 2C19, 2D6, and 3A4/5 in human liver microsomal preparations from livers L1 to L12 were determined by quantitative Western blotting In Vitro Metabolic Incubations and Metabolite Analysis. Incubations of index substrates with human liver microsomes and lymphoblas

Quantitative Prediction and Clinical Observation of a CYP3A Inhibitor-Based Drug-Drug Interactions with MLN3897, a Potent C-C Chemokine Receptor-1 Antagonist

ABSTRACT A novel in vitro model was recently developed in our laboratories for the prediction of magnitude of clinical pharmacokinetic drugdrug interactions (DDIs), based on reversible hepatic cytochrome P450 (P450) inhibition. This approach, using inhibition data from human hepatocytes incubated in human plasma, and quantitative P450 phenotyping data from hepatic microsomal incubations, successfully predicted DDIs for 15 marketed drugs with ketoconazole, a strong competitive inhibitor of CYP3A4/5, generally used to demonstrate a "worst-case scenario" for CYP3A inhibition. In addition, this approach was successfully extended to DDI predictions with the moderate competitive CYP3A inhibitor fluconazole for nine marketed drugs. In the current report, the general applicability of the model has been demonstrated by prospectively predicting the degree of inhibition and then conducting DDI studies in the clinic for an investigational CCR1 antagonist MLN3897, which is cleared predominantly by CYP3A. The clinical studies involved treatment of healthy volunteers (n ϭ 17-20), in a crossover design, with ketoconazole (200 mg b.i.d.) or fluconazole (400 mg once a day), while receiving MLN3897. Administration of MLN3897 and ketoconazole led to an average 8.28-fold increase in area under the curve of plasma concentration-time plot (AUC) of MLN3897 at steady state, compared with the 8.33-fold increase predicted from the in vitro data. Similarly for fluconazole, an average increase of 3.93-fold in AUC was observed for MLN3897 in comparison with a predicted value of 3.26-fold. Thus, our model reliably predicted the exposure changes for MLN3897 in interaction studies with competitive CYP3A inhibitors in humans, further strengthening the utility of our in vitro model. Prediction of clinical DDIs using in vitro studies is one of the major challenges in the pharmaceutical industry. The main utility of such DDI predictions is to help foresee any safety issues anticipated from higher exposures and thus help design clinical trials with better safety. In some instances, clinical DDI studies can be avoided if no significant pharmacokinetic interaction is predicted. Traditionally, DDI predictions have been based on the ratio of the inhibitor concentration [I] and the enzyme inhibition constant K i ([I]/K i ratio

A survey of across-target bioactivity results of small molecules in PubChem

This work provides an analysis of across-target bioactivity results in the screening data deposited in PubChem. Two alternative approaches for grouping-related targets are used to examine a compound's across-target bioactivity. This analysis identifies compounds that are selectively active against groups of protein targets that are identical or similar in sequence. This analysis also identifies compounds that are bioactive across unrelated targets. Statistical distributions of compound' across-target selectivity provide a survey to evaluate target specificity of compounds by deriving and analyzing bioactivity profile across a wide range of biological targets for tested small molecules in PubChem. This work enables one to select target specific inhibitors, identify promiscuous compounds and better understand the biological mechanisms of target-small molecule interactions

Bile acid–sensitive tuft cells regulate biliary neutrophil influx

Inflammation and dysfunction of the extrahepatic biliary tree are common causes of human pathology, including gallstones and cholangiocarcinoma. Despite this, we know little about the local regulation of biliary inflammation. Tuft cells, rare sensory epithelial cells, are particularly prevalent in the mucosa of the gallbladder and extrahepatic bile ducts. Here, we show that biliary tuft cells express a core genetic tuft cell program in addition to a tissue-specific gene signature and, in contrast to small intestinal tuft cells, decreased postnatally, coincident with maturation of bile acid production. Manipulation of enterohepatic bile acid recirculation revealed that tuft cell abundance is negatively regulated by bile acids, including in a model of obstructive cholestasis in which inflammatory infiltration of the biliary tree correlated with loss of tuft cells. Unexpectedly, tuft cell–deficient mice spontaneously displayed an increased gallbladder epithelial inflammatory gene signature accompanied by neutrophil infiltration that was modulated by the microbiome. We propose that biliary tuft cells function as bile acid–sensitive negative regulators of inflammation in biliary tissues and serve to limit inflammation under homeostatic conditions

SER-109: An Oral Investigational Microbiome Therapeutic for Patients with Recurrent Clostridioides difficile Infection (rCDI)

Clostridioides difficile infection (CDI) is classified as an urgent health threat by the Centers for Disease Control and Prevention (CDC), and affects nearly 500,000 Americans annually. Approximately 20–25% of patients with a primary infection experience a recurrence, and the risk of recurrence increases with subsequent episodes to greater than 40%. The leading risk factor for CDI is broad-spectrum antibiotics, which leads to a loss of microbial diversity and impaired colonization resistance. Current FDA-approved CDI treatment strategies target toxin or toxin-producing bacteria, but do not address microbiome disruption, which is key to the pathogenesis of recurrent CDI. Fecal microbiota transplantation (FMT) reduces the risk of recurrent CDI through the restoration of microbial diversity. However, FDA safety alerts describing hospitalizations and deaths related to pathogen transmission have raised safety concerns with the use of unregulated and unstandardized donor-derived products. SER-109 is an investigational oral microbiome therapeutic composed of purified spore-forming Firmicutes. SER-109 was superior to a placebo in reducing CDI recurrence at Week 8 (12% vs. 40%, respectively; p \u3c 0.001) in adults with a history of recurrent CDI with a favorable observed safety profile. Here, we discuss the role of the microbiome in CDI pathogenesis and the clinical development of SER-109, including its rigorous manufacturing process, which mitigates the risk of pathogen transmission. Additionally, we discuss compositional and functional changes in the gastrointestinal microbiome in patients with recurrent CDI following treatment with SER-109 that are critical to a sustained clinical response

Safety and Tolerability of SER-109 as an Investigational Microbiome Therapeutic in Adults With Recurrent Clostridioides difficile Infection: A Phase 3, Open-Label, Single-Arm Trial

IMPORTANCE: A safe and effective treatment for recurrent Clostridioides difficile infection (CDI) is urgently needed. Antibiotics kill toxin-producing bacteria but do not repair the disrupted microbiome, which promotes spore germination and infection recurrence. OBJECTIVES: To evaluate the safety and rate of CDI recurrence after administration of investigational microbiome therapeutic SER-109 through 24 weeks. DESIGN, SETTING, AND PARTICIPANTS: This phase 3, single-arm, open-label trial (ECOSPOR IV) was conducted at 72 US and Canadian outpatient sites from October 2017 to April 2022. Adults aged 18 years or older with recurrent CDI were enrolled in 2 cohorts: (1) rollover patients from the ECOSPOR III trial who had CDI recurrence diagnosed by toxin enzyme immunoassay (EIA) and (2) patients with at least 1 CDI recurrence (diagnosed by polymerase chain reaction [PCR] or toxin EIA), inclusive of their acute infection at study entry. INTERVENTIONS: SER-109 given orally as 4 capsules daily for 3 days following symptom resolution after antibiotic treatment for CDI. MAIN OUTCOMES AND MEASURES: The main outcomes were safety, measured as the rate of treatment-emergent adverse events (TEAEs) in all patients receiving any amount of SER-109, and cumulative rates of recurrent CDI (toxin-positive diarrhea requiring treatment) through week 24 in the intent-to-treat population. RESULTS: Of 351 patients screened, 263 were enrolled (180 [68.4%] female; mean [SD] age, 64.0 [15.7] years); 29 were in cohort 1 and 234 in cohort 2. Seventy-seven patients (29.3%) were enrolled with their first CDI recurrence. Overall, 141 patients (53.6%) had TEAEs, which were mostly mild to moderate and gastrointestinal. There were 8 deaths (3.0%) and 33 patients (12.5%) with serious TEAEs; none were considered treatment related by the investigators. Overall, 23 patients (8.7%; 95% CI, 5.6%-12.8%) had recurrent CDI at week 8 (4 of 29 [13.8%; 95% CI, 3.9%-31.7%] in cohort 1 and 19 of 234 [8.1%; 95% CI, 5.0%-12.4%] in cohort 2), and recurrent CDI rates remained low through 24 weeks (36 patients [13.7%; 95% CI, 9.8%-18.4%]). At week 8, recurrent CDI rates in patients with a first recurrence were similarly low (5 of 77 [6.5%; 95% CI, 2.1%-14.5%]) as in patients with 2 or more recurrences (18 of 186 [9.7%; 95% CI, 5.8%-14.9%]). Analyses by select baseline characteristics showed consistently low recurrent CDI rates in patients younger than 65 years vs 65 years or older (5 of 126 [4.0%; 95% CI, 1.3%-9.0%] vs 18 of 137 [13.1%; 95% CI, 8.0%-20.0%]) and patients enrolled based on positive PCR results (3 of 69 [4.3%; 95% CI, 0.9%-12.2%]) vs those with positive toxin EIA results (20 of 192 [10.4%; 95% CI, 6.5%-15.6%]). CONCLUSIONS AND RELEVANCE: In this trial, oral SER-109 was well tolerated in a patient population with recurrent CDI and prevalent comorbidities. The rate of recurrent CDI was low regardless of the number of prior recurrences, demographics, or diagnostic approach, supporting the beneficial impact of SER-109 for patients with CDI. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03183141

Diclofenac Prolongs Repolarization in Ventricular Muscle with Impaired Repolarization Reserve

Background: The aim of the present work was to characterize the electrophysiological effects of the non-steroidal anti- inflammatory drug diclofenac and to study the possible proarrhythmic potency of the drug in ventricular muscle. Methods: Ion currents were recorded using voltage clamp technique in canine single ventricular cells and action potentials were obtained from canine ventricular preparations using microelectrodes. The proarrhythmic potency of the drug was investigated in an anaesthetized rabbit proarrhythmia model. Results: Action potentials were slightly lengthened in ventricular muscle but were shortened in Purkinje fibers by diclofenac (20 mM). The maximum upstroke velocity was decreased in both preparations. Larger repolarization prolongation was observed when repolarization reserve was impaired by previous BaCl 2 application. Diclofenac (3 mg/kg) did not prolong while dofetilide (25 mg/kg) significantly lengthened the QT c interval in anaesthetized rabbits. The addition of diclofenac following reduction of repolarization reserve by dofetilide further prolonged QT c . Diclofenac alone did not induce Torsades de Pointes ventricular tachycardia (TdP) while TdP incidence following dofetilide was 20%. However, the combination of diclofenac and dofetilide significantly increased TdP incidence (62%). In single ventricular cells diclofenac (30 mM) decreased the amplitude of rapid (I Kr ) and slow (I Ks ) delayed rectifier currents thereby attenuating repolarization reserve. L-type calcium current (I Ca ) was slightly diminished, but the transient outward (I to ) and inward rectifier (I K1 ) potassium currents were not influenced. Conclusions: Diclofenac at therapeutic concentrations and even at high dose does not prolong repolarization markedly and does not increase the risk of arrhythmia in normal heart. However, high dose diclofenac treatment may lengthen repolarization and enhance proarrhythmic risk in hearts with reduced repolarization reserve

Safety and Tolerability of SER-109 as an Investigational Microbiome Therapeutic in Adults With Recurrent Clostridioides difficile Infection: A Phase 3, Open-Label, Single-Arm Trial

IMPORTANCE: A safe and effective treatment for recurrent Clostridioides difficile infection (CDI) is urgently needed. Antibiotics kill toxin-producing bacteria but do not repair the disrupted microbiome, which promotes spore germination and infection recurrence. OBJECTIVES: To evaluate the safety and rate of CDI recurrence after administration of investigational microbiome therapeutic SER-109 through 24 weeks. DESIGN, SETTING, AND PARTICIPANTS: This phase 3, single-arm, open-label trial (ECOSPOR IV) was conducted at 72 US and Canadian outpatient sites from October 2017 to April 2022. Adults aged 18 years or older with recurrent CDI were enrolled in 2 cohorts: (1) rollover patients from the ECOSPOR III trial who had CDI recurrence diagnosed by toxin enzyme immunoassay (EIA) and (2) patients with at least 1 CDI recurrence (diagnosed by polymerase chain reaction [PCR] or toxin EIA), inclusive of their acute infection at study entry. INTERVENTIONS: SER-109 given orally as 4 capsules daily for 3 days following symptom resolution after antibiotic treatment for CDI. MAIN OUTCOMES AND MEASURES: The main outcomes were safety, measured as the rate of treatment-emergent adverse events (TEAEs) in all patients receiving any amount of SER-109, and cumulative rates of recurrent CDI (toxin-positive diarrhea requiring treatment) through week 24 in the intent-to-treat population. RESULTS: Of 351 patients screened, 263 were enrolled (180 [68.4%] female; mean [SD] age, 64.0 [15.7] years); 29 were in cohort 1 and 234 in cohort 2. Seventy-seven patients (29.3%) were enrolled with their first CDI recurrence. Overall, 141 patients (53.6%) had TEAEs, which were mostly mild to moderate and gastrointestinal. There were 8 deaths (3.0%) and 33 patients (12.5%) with serious TEAEs; none were considered treatment related by the investigators. Overall, 23 patients (8.7%; 95% CI, 5.6%-12.8%) had recurrent CDI at week 8 (4 of 29 [13.8%; 95% CI, 3.9%-31.7%] in cohort 1 and 19 of 234 [8.1%; 95% CI, 5.0%-12.4%] in cohort 2), and recurrent CDI rates remained low through 24 weeks (36 patients [13.7%; 95% CI, 9.8%-18.4%]). At week 8, recurrent CDI rates in patients with a first recurrence were similarly low (5 of 77 [6.5%; 95% CI, 2.1%-14.5%]) as in patients with 2 or more recurrences (18 of 186 [9.7%; 95% CI, 5.8%-14.9%]). Analyses by select baseline characteristics showed consistently low recurrent CDI rates in patients younger than 65 years vs 65 years or older (5 of 126 [4.0%; 95% CI, 1.3%-9.0%] vs 18 of 137 [13.1%; 95% CI, 8.0%-20.0%]) and patients enrolled based on positive PCR results (3 of 69 [4.3%; 95% CI, 0.9%-12.2%]) vs those with positive toxin EIA results (20 of 192 [10.4%; 95% CI, 6.5%-15.6%]). CONCLUSIONS AND RELEVANCE: In this trial, oral SER-109 was well tolerated in a patient population with recurrent CDI and prevalent comorbidities. The rate of recurrent CDI was low regardless of the number of prior recurrences, demographics, or diagnostic approach, supporting the beneficial impact of SER-109 for patients with CDI. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03183141