Pharmacokinetics of asundexian with combined CYP3A and P-gp inhibitors and an inducer: Target in vitro and in vivo studies.

AIMS: Asundexian is an oral, direct and reversible inhibitor of activated factor XI (FXIa) in development for the treatment of thromboembolic events. This article summarizes results from preclinical and clinical studies, including identification of enzymes involved in asundexian pharmacokinetics, and evaluation of potential target drug-drug interactions. METHODS: In vitro studies investigated the substrate characteristics of asundexian towards several cytochrome P450 (CYP) isoforms, hydrolytic enzymes and drug transporters. Inhibition of the amide hydrolysis of asundexian was investigated in vitro for several relevant drugs. Phase 1 studies in healthy male participants investigated the pharmacokinetics (PK) of asundexian upon co-administration of combined inhibitors or an inducer of P-gp and CYP3A4 (itraconazole, verapamil or carbamazepine, respectively, or the moderate CYP3A4 inhibitor fluconazole). The pharmacodynamic (PD) markers are activated partial thromboplastin time and FXIa inhibition. RESULTS: Asundexian was predominantly metabolized via carboxylesterase 1 and, to a lesser extent, via CYP3A4 and is a substrate of P-gp. The asundexian area under the plasma concentration-time curve (AUC) increased by 103% and 75.6% upon combined inhibition of P-gp and strong or moderate inhibition of CYP3A4, respectively, but was unaffected by moderate CYP3A4 inhibition. Combined P-gp and CYP3A4 induction by carbamazepine decreased asundexian AUC by 44.4%. PD is concentration-dependent, thus no differences in maximum responses and recovery commensurate with PK effect(s) were observed. Adverse events were mild and asundexian was well tolerated. CONCLUSIONS: The presented studies confirmed that CYP3A4 and P-gp contribute to asundexian metabolism and excretion. Observed effects were in line with data from a previous mass balance study.

In vivo Metabolism of Nifurtimox and the Drug-Drug Interaction Potential Including its Major Metabolites.

BACKGROUND: Nifurtimox is an effective treatment for patients with Chagas disease, but knowledge of its biotransformation and excretion is limited. OBJECTIVE: This study aimed to better understand the fate of oral nifurtimox in vivo. METHODS: We investigated the exposure and excretion pathways of [14C]-labeled nifurtimox and its metabolites in rats. We then quantified the prominent metabolites and nifurtimox in the urine and plasma of patients receiving nifurtimox using LC-HRMS with reference standards and quantified these compounds in rat plasma after a single, high dose of nifurtimox. We also investigated potential drug-drug interactions (DDIs) of these compounds in vitro. RESULTS: In rats, orally administered nifurtimox was rapidly absorbed (tmax 0.5 h) and eliminated (t½ 1.4 h). Metabolism of nifurtimox yielded six predominant metabolites (M-1 to M-6) in urine and plasma, and the dose was excreted equally via the renal and fecal routes with only traces of unchanged nifurtimox detectable due to its instability in excreta. In patients with Chagas disease, only M-6 and M-4 achieved relevant exposure levels, and the total amount of excreted metabolites in urine was higher in fed versus fasted patients, consistent with the higher systemic exposure. For nifurtimox, M-6, and M-4, no potential perpetrator pharmacokinetic DDIs with the main cytochrome P- 450 enzymes and drug transporters were identified in vitro. CONCLUSION: This contemporary analysis of the complex metabolite profile and associated exposures emerging after oral dosing of nifurtimox in rats and humans, together with the expected low risk for clinically relevant DDIs, expands the understanding of this important anti-trypanosomal drug.

Results From Drug-Drug Interaction Studies In Vitro and In Vivo Investigating the Inhibitory Effect of Finerenone on the Drug Transporters BCRP, OATP1B1, and OATP1B3.

BACKGROUND AND OBJECTIVES: In vitro and in vivo studies were performed with the novel, selective, nonsteroidal mineralocorticoid receptor antagonist finerenone to assess the relevance of inhibitory effects on the transporters breast cancer resistance protein (BCRP), organic anion transporting polypeptide 1B1 (OATP1B1), and OATP1B3. These transporters are involved in the disposition of a number of drugs, including statins. Statins are also a frequent comedication in patients receiving finerenone. Therefore, inhibitory effects on BCRP and OATPs are of potential clinical relevance. METHODS: The effect on the transport of specific substrates of BCRP and OATP1B1/1B3 was assessed in cell-based in vitro assays with finerenone or its metabolites. A fixed-sequence crossover study in 14 healthy male volunteers investigated the effects of finerenone (40 mg once daily) on the pharmacokinetics of the index substrate rosuvastatin (5 mg) administered alone, simultaneously with, or approximately 4 h before finerenone. The effect of finerenone on the endogenous OATP substrates coproporphyrin I and III was also assessed. RESULTS: Based on in vitro findings and threshold values proposed in regulatory guidelines, finerenone appeared to be a potentially relevant inhibitor of all three transporters. Relevant inhibition could also not be ruled out for the finerenone metabolites M1a (OATP1B1) and M3a (OATP1B1 and OAT1B3), which prompted an investigation into the relevance of these findings in vivo. After administration on a background of finerenone 40 mg, all point estimates of area under the curve ratios (114.47% [rosuvastatin], 99.62% [coproporphyrin I; simultaneous], and 105.28% [rosuvastatin; 4 h separation]) and maximum concentration ratios (111.24% [rosuvastatin], 101.22% [coproporphyrin I], 89.14% [coproporphyrin III; simultaneous], and 96.84% [rosuvastatin; 4 h separation]) of the investigated substrates were within 80.0-125%. In addition, the 90% confidence intervals of the ratios were within the conventional no-effect boundaries of 80.0% and 125% for rosuvastatin after temporally separated administration, and for coproporphyrin I and III. CONCLUSION: Administration of finerenone 40 mg once daily confers no risk of clinically relevant drug-drug interactions with substrates of BCRP, OATP1B1, or OATP1B3. The potential for relevant inhibition of these transporters suggested by in vitro findings was not confirmed in vivo.

Finerenone is a drug that is used to reduce the risk of adverse kidney and cardiovascular outcomes in patients with chronic kidney disease associated with type 2 diabetes mellitus. Taking more than one medicine, and often several at the same time, is common in these patients; therefore, it is important to investigate the drug­drug interaction potential of finerenone. These studies were carried out to assess the interaction potential of finerenone and its metabolites in both laboratory experiments and healthy volunteers. Initial laboratory experiments indicated that finerenone and its metabolites could inhibit transporters used by other drugs. A study in healthy volunteers was performed and demonstrated that finerenone is not associated with any clinically meaningful changes to drugs that are substrates for the transporters. The study in healthy volunteers demonstrates that medications that are substrates of these transporters can be safely co-administered with finerenone.

The Effects of Weak and Strong CYP3A Induction by Rifampicin on the Pharmacokinetics of Five Progestins and Ethinylestradiol Compared to Midazolam.

It is known that co-administration of CYP3A inducers may decrease the effectiveness of oral contraceptives containing progestins as mono-preparations or combined with ethinylestradiol. In a randomized clinical drug-drug interaction study, we investigated the effects of CYP3A induction on the pharmacokinetics of commonly used progestins and ethinylestradiol. Rifampicin was used to induce CYP3A. The progestins chosen as victim drugs were levonorgestrel, norethindrone, desogestrel, and dienogest as mono-products, and drospirenone combined with ethinylestradiol. Postmenopausal women (n = 12-14 per treatment group) received, in fixed sequence, a single dose of the victim drug plus midazolam without rifampicin, with rifampicin 10 mg/day (weak induction), and with rifampicin 600 mg/day (strong induction). The effects on progestin exposure were compared with the effects on midazolam exposure (as a benchmark). Unbound concentrations were evaluated for drugs binding to sex hormone binding globulin. Weak CYP3A induction, as confirmed by a mean decrease in midazolam exposure by 46%, resulted in minor changes in progestin exposure (mean decreases: 15-37%). Strong CYP3A induction, in contrast, resulted in mean decreases by 57-90% (mean decrease in midazolam exposure: 86%). Namely, the magnitude of the observed induction effects varied from weak to strong. Our data might provide an impetus to revisit the currently applied clinical recommendations for oral contraceptives, especially for levonorgestrel and norethindrone-containing products, and they might give an indication as to which progestin could be used, if requested, by women taking weak CYP3A inducers-although it is acknowledged that the exact exposure-response relationship for contraceptive efficacy is currently unclear for most progestins.

Evaluation of Clinically Relevant Drug-Drug Interactions and Population Pharmacokinetics of Darolutamide in Patients with Nonmetastatic Castration-Resistant Prostate Cancer: Results of Pre-Specified and Post Hoc Analyses of the Phase III ARAMIS Trial.

BACKGROUND: Darolutamide, an androgen receptor antagonist with a distinct molecular structure, significantly prolonged metastasis-free survival versus placebo in the phase III ARAMIS study in men with nonmetastatic castration-resistant prostate cancer (nmCRPC). In this population, polypharmacy for age-related comorbidities is common and may increase drug-drug interaction (DDI) risks. Preclinical/phase I study data suggest darolutamide has a low DDI potential-other than breast cancer resistance protein/organic anion transporter protein substrates (e.g., statins), no clinically relevant effect on comedications is expected. OBJECTIVE: Our objective was to evaluate the effect of commonly administered drugs on the pharmacokinetics of darolutamide and the effect of comedications potentially affected by darolutamide on safety in patients with nmCRPC. PATIENTS AND METHODS: Comorbidities and comedication use in the 1509 ARAMIS participants treated with darolutamide 600 mg twice daily or placebo were assessed. A population pharmacokinetic analysis evaluated whether comedications affected the pharmacokinetics of darolutamide in a subset of 388 patients. A subgroup analysis of adverse events (AEs) in statin users versus nonusers was conducted. RESULTS: Most participants (median age 74 years) had at least one comorbidity (98.4% in both arms) and used at least one comedication (98.7% with darolutamide vs. 98.0% with placebo); these were similar across study arms. Despite frequent use of comedications with DDI potential, no significant effects on darolutamide pharmacokinetics were identified. Comedications included lipid-modifying agents (34.5%), ß-blockers (29.7%), antithrombotics (42.8%), and systemic antibiotics (26.9%). AE incidence was similar across study arms in statin users and nonusers. Study limitations include the small sample size for sub-analyses. CONCLUSIONS: These analyses suggest the pharmacokinetic profile of darolutamide is not affected by a number of commonly administered drugs in patients with nmCRPC. Although pharmacokinetic data have indicated that darolutamide has the potential to interact with rosuvastatin, used to assess DDI in these studies, this finding did not seem to translate into increased AEs due to statin use in the ARAMIS trial. Clinicaltrials.gov identifier: NCT02200614.

BACKGROUND: Darolutamide is a medicine used to treat men with prostate cancer that has not spread to other parts of the body (nonmetastatic). Often, these patients are taking other medicines for common age-related illnesses. Taking more than one medicine at the same time increases the chances of what is known as drug­drug interactions. Drug­drug interactions can decrease how well the medicines work or may sometimes increase side effects. STUDY AIM: To test for possible drug­drug interactions in men with prostate cancer who take darolutamide alongside other medicines. STUDY PARTICIPANTS: Men with nonmetastatic prostate cancer who were being treated with a medicine that lowers testosterone, a chemical in the body that causes prostate cancer tumors to grow. Participants took two darolutamide 300 mg tablets, or an inactive placebo, twice a day. WHAT DID THE RESEARCHERS MEASURE?: The researchers documented the number of medicines taken by each participant and the number of other medical conditions that they had. Tests were done to find out whether other medicines affected the way that darolutamide works in the body and whether patients taking darolutamide alongside other medicines experienced more side effects. RESULTS: As would be expected, based on the typical age of patients with prostate cancer, more than 90% of participants in this study used medicines other than darolutamide to manage common age-related illnesses or medical conditions. Taking medicines alongside darolutamide did not impact how darolutamide worked in the body and did not increase the number of side effects experienced by patients. Darolutamide is known to interact with rosuvastatin, a cholesterol-lowering drug. However, in this study, there was no overall increase in side effects among darolutamide-treated patients who took this type of drug compared with in those who did not. CONCLUSION: In this study of patients with nonmetastatic prostate cancer, limited drug­drug interactions were seen when taking darolutamide alongside other medicines given to these patients to manage age-related medical conditions.

Drug-Drug Interaction Potential of Darolutamide: In Vitro and Clinical Studies.

BACKGROUND AND OBJECTIVES: Darolutamide is a novel androgen receptor (AR) antagonist approved for the treatment of nonmetastatic castration-resistant prostate cancer (nmCRPC). Accordingly, the drug-drug interaction (DDI) potential of darolutamide was investigated in both nonclinical and clinical studies. METHODS: In vitro studies were performed to determine the potential for darolutamide to be a substrate, inducer or inhibitor for cytochrome P450 (CYP) isoforms, other metabolizing enzymes and drug transporters. A phase I drug-interaction study in healthy volunteers evaluated the impact of co-administering rifampicin [CYP3A4 and P-glycoprotein (P-gp) inducer] and itraconazole [CYP3A4, P-gp and breast cancer resistance protein (BCRP) inhibitor] on the pharmacokinetics of darolutamide. Two further phase I studies assessed the impact of co-administering oral darolutamide on the pharmacokinetics of midazolam (sensitive CYP3A4 substrate) and dabigatran etexilate (P-gp substrate) and the impact on the pharmacokinetics of co-administered rosuvastatin [a substrate for BCRP, organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and organic anion transporter (OAT)3]. RESULTS: In vitro, darolutamide was predominantly metabolized via oxidative biotransformation catalyzed by CYP3A4 and was identified as a substrate for P-gp and BCRP. The enzymatic activity of nine CYP isoforms was not inhibited or slightly inhibited in vitro with darolutamide, and a rank order and mechanistic static assessment indicated that risk of clinically relevant DDIs via CYP inhibition is very low. In vitro, darolutamide exhibited no relevant induction of CYP1A2 or CYP2B6 activity. Inhibition of BCRP-, P-gp-, OAT3-, MATE1-, MATE2-K-, OATP1B1- and OATP1B3-mediated transport was observed in vitro. Phase I data showed that darolutamide exposure increased 1.75-fold with co-administered itraconazole and decreased by 72% with rifampicin. Co-administration of darolutamide with CYP3A4/P-gp substrates showed no effect or only minor effects. Rosuvastatin exposure increased 5.2-fold with darolutamide because of BCRP and probably also OATPB1/OATPB3 inhibition. CONCLUSIONS: Darolutamide has a low potential for clinically relevant DDIs with drugs that are substrates for CYP or P-gp; increased exposure of BCRP and probably OATP substrates was the main interaction of note.

Industry Perspective on Contemporary Protein-Binding Methodologies: Considerations for Regulatory Drug-Drug Interaction and Related Guidelines on Highly Bound Drugs.

Regulatory agencies have recently issued drug-drug interaction guidelines, which require determination of plasma protein binding (PPB). To err on the conservative side, the agencies recommend that a 0.01 lower limit of fraction unbound (fu) be used for highly bound compounds (>99%), irrespective of the actual measured values. While this may avoid false negatives, the recommendation would likely result in a high rate of false positive predictions, resulting in unnecessary clinical studies and more stringent inclusion/exclusion criteria, which may add cost and time in delivery of new medicines to patients. In this perspective, we provide a review of current approaches to measure PPB, and important determinants in enabling the accuracy and precision in these measurements. The ability to measure fu is further illustrated by a cross-company data comparison of PPB for warfarin and itraconazole, demonstrating good concordance of the measured fu values. The data indicate that fu values of ≤0.01 may be determined accurately across laboratories when appropriate methods are used. These data, along with numerous other examples presented in the literature, support the use of experimentally measured fu values for drug-drug interaction predictions, rather than using the arbitrary cutoff value of 0.01 as recommended in current regulatory guidelines.

Imaging of Sarcoidosis: A Contemporary Review.

Sarcoidosis is a systemic granulomatous disorder with a variety of clinical presentations and radiological appearances. Although it primarily affects the lungs and lymphatics, sarcoidosis potentially involves essentially every organ system. On imaging, sarcoidosis can mimic different disease entities, including primary and metastatic neoplasms, vasculitis, and other granulomatous infections. Definitive diagnosis often requires a combination of clinical, radiological, and histologic information. Imaging plays a crucial role in diagnosis and evaluating response to therapy. This review covers imaging findings in sarcoidosis within each organ system, with an emphasis on the use of imaging in the diagnosis and management of this condition.

Dysregulation of presynaptic calcium and synaptic plasticity in a mouse model of 22q11 deletion syndrome.

The 22q11 deletion syndrome (22q11DS) is characterized by cognitive decline and increased risk of psychiatric disorders, mainly schizophrenia. The molecular mechanisms of neuronal dysfunction in cognitive symptoms of 22q11DS are poorly understood. Here, we report that a mouse model of 22q11DS, the Df(16)1/+ mouse, exhibits substantially enhanced short- and long-term synaptic plasticity at hippocampal CA3-CA1 synapses, which coincides with deficits in hippocampus-dependent spatial memory. These changes are evident in mature but not young animals. Electrophysiological, two-photon imaging and glutamate uncaging, and electron microscopic assays in acute brain slices showed that enhanced neurotransmitter release but not altered postsynaptic function or structure caused these changes. Enhanced neurotransmitter release in Df(16)1/+ mice coincided with altered calcium kinetics in CA3 presynaptic terminals and upregulated sarco(endo)plasmic reticulum calcium-ATPase type 2 (SERCA2). SERCA inhibitors rescued synaptic phenotypes of Df(16)1/+ mice. Thus, presynaptic SERCA2 upregulation may be a pathogenic event contributing to the cognitive symptoms of 22q11DS.

The F-BAR protein family Actin' on the membrane.

A tight spatio-temporal coordination of the machineries controlling actin dynamics and membrane remodelling is crucial for a huge variety of cellular processes that shape cells into a multicellular organism. Dynamic membrane remodelling is achieved by a functional relationship between proteins that control plasma membrane curvature, membrane fission and nucleation of new actin filaments. The BAR/F-BAR-domain-containing proteins are prime candidates to couple plasma membrane curvature and actin dynamics in different morphogenetic processes. Here, we discuss recent findings on the membrane-shaping proteins of the F-BAR domain subfamily and how they regulate morphogenetic processes in vivo.

Drosophila Cip4/Toca-1 integrates membrane trafficking and actin dynamics through WASP and SCAR/WAVE.

BACKGROUND: Developmental processes are intimately tied to signaling events that integrate the dynamic reorganization of the actin cytoskeleton and membrane dynamics. The F-BAR-domain-containing proteins are prime candidates to couple actin dynamics and membrane trafficking in different morphogenetic processes. RESULTS: Here, we present the functional analysis of the Drosophila F-BAR protein Cip4/Toca1 (Cdc42-interacting protein 4/transducer of Cdc42-dependent actin assembly 1). Cip4 is able to form a complex with WASP and SCAR/WAVE and recruits both actin-nucleation-promoting factors to invaginating membranes and endocytic vesicles. Actin-comet-tail-based movement of these vesicles depends not only on WASP but largely on WAVE function. In vivo, loss of cip4 function causes multiple wing hairs. A similar phenotype is observed when vesicle scission is affected after Dynamin suppression. Gene dosage experiments show that Cip4 and WAVE functionally interact to restrict wing hair formation. Further rescue experiments confirm that Cip4 is able to act through WAVE and WASP in vivo. Biochemical and functional data support a model in which Cdc42 acts upstream of Cip4 and recruits not only WASP but also SCAR/WAVE via Abi to control Dynamin-dependent cell polarization in the wing. CONCLUSION: Cip4 integrates membrane trafficking and actin dynamics through WASP and WAVE. First, Cip4 promotes membrane invaginations and triggers the vesicle scission by recruiting Dynamin to the neck of nascent vesicles. Second, Cip4 recruits WASP and WAVE proteins to induce actin polymerization, supporting vesicle scission and providing the force for vesicle movement.

Syndapin promotes formation of a postsynaptic membrane system in Drosophila.

Syndapins belong to the F-BAR domain protein family whose predicted functions in membrane tubulation remain poorly studied in vivo. At Drosophila neuromuscular junctions, syndapin is associated predominantly with a tubulolamellar postsynaptic membrane system known as the subsynaptic reticulum (SSR). We show that syndapin overexpression greatly expands this postsynaptic membrane system. Syndapin can expand the SSR in the absence of dPAK and Dlg, two known regulators of SSR development. Syndapin's N-terminal F-BAR domain, required for membrane tubulation in cultured cells, is required for SSR expansion. Consistent with a model in which syndapin acts directly on postsynaptic membrane, SSR expansion requires conserved residues essential for membrane binding in vitro. However, syndapin's Src homology (SH) 3 domain, which negatively regulates membrane tubulation in cultured cells, is required for synaptic targeting and strong SSR induction. Our observations advance knowledge of syndapin protein function by 1) demonstrating the in vivo relevance of membrane remodeling mechanisms suggested by previous in vitro and structural analyses, 2) showing that SH3 domains are necessary for membrane expansion observed in vivo, and 3) confirming that F-BAR proteins control complex membrane structures.

Drosophila Cip4 and WASp define a branch of the Cdc42-Par6-aPKC pathway regulating E-cadherin endocytosis.

BACKGROUND: Integral to the function and morphology of the epithelium is the lattice of cell-cell junctions known as adherens junctions (AJs). AJ stability and plasticity relies on E-Cadherin exocytosis and endocytosis. A mechanism regulating E-Cadherin (E-Cad) exocytosis to the AJs has implicated proteins of the exocyst complex, but mechanisms regulating E-Cad endocytosis from the AJs remain less well understood. RESULTS: Here we show that Cdc42, Par6, or aPKC loss of function is accompanied by the accumulation of apical E-Cad intracellular punctate structures and the disruption of AJs in Drosophila epithelial cells. These punctate structures derive from large and malformed endocytic vesicles that emanate from the AJs; a phenotype that is also observed upon blocking vesicle scission in dynamin mutant cells. We demonstrate that the Drosophila Cdc42-interacting protein 4 (Cip4) is a Cdc42 effector that interacts with Dynamin and the Arp2/3 activator WASp in Drosophila. Accordingly, Cip4, WASp, or Arp2/3 loss of function also results in defective E-Cadherin endocytosis. CONCLUSION: Altogether our results show that Cdc42 functions with Par6 and aPKC to regulate E-Cad endocytosis and define Cip4 and WASp as regulators of the early E-Cad endocytic events in epithelial tissue.

Slow presynaptic and fast postsynaptic components of compound long-term potentiation.

Long-term potentiation (LTP) mediates learning and memory in the mammalian hippocampus. Whether a presynaptic or postsynaptic neuron principally enhances synaptic transmission during LTP remains controversial. Acute hippocampal slices were made from transgenic mouse strains that express synaptopHluorin in neurons. SynaptopHluorin is an indicator of synaptic vesicle recycling; thus, we monitored functional changes in presynaptic boutons of CA3 pyramidal cells by measuring changes in synaptopHluorin fluorescence. Simultaneously, we recorded field excitatory postsynaptic potentials to monitor changes in the strength of excitatory synapses between CA3 and CA1 pyramidal neurons. We found that LTP consists of two components, a slow presynaptic component and a fast postsynaptic component. The presynaptic mechanisms contribute mostly to the late phase of compound LTP, whereas the postsynaptic mechanisms are crucial during the early phase of LTP. We also found that protein kinase A (PKA) and L-type voltage-gated calcium channels are crucial for the expression of the presynaptic component of compound LTP, and NMDA channels are essential for that of the postsynaptic component of LTP. These data are the first direct evidence that presynaptic and postsynaptic components of LTP are temporally and mechanistically distinct.