Evaluation of the pharmacokinetics of pemigatinib in patients with impaired hepatic or renal function.

AIMS: Pemigatinib, an inhibitor of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases, is approved for previously treated, unresectable locally advanced or metastatic cholangiocarcinoma. Pemigatinib is predominantly metabolized by CYP3A4 with minimal renal elimination. METHODS: Separate hepatic and renal impairment studies were conducted to evaluate the effect of these impairments on pemigatinib pharmacokinetics (PK). Each study was of open-label, parallel-group design, conducted in participants with normal organ function and with hepatic or renal impairment. Plasma concentrations of pemigatinib were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) and pemigatinib PK parameters were derived by noncompartmental analysis. Geometric mean ratios and two-sided 90% confidence intervals of Cmax , AUC0-t , and AUC0-∞ were compared by analysis of variance (ANOVA). RESULTS: Compared with healthy matched participants: Cmax and AUC0-∞ ratio (90% confidence interval) in participants with moderate hepatic impairment were 96.7% (59.4%, 157%) and 146% (100%, 212%), respectively; Cmax and AUC0-∞ ratio in participants with severe hepatic impairment were 94.2% (68.9%, 129%) and 174% (116%, 261%), respectively; Cmax and AUC0-∞ ratio in participants with severe renal impairment were 64.6% (44.1%, 94.4%) and 159% (95.4%, 264%), respectively; Cmax and AUC0-∞ ratio in participants with end-stage renal disease (ESRD) before haemodialysis (HD) were 77.5% (51.2%, 118%) and 76.8% (54.0%, 109%), respectively; Cmax and AUC0-∞ ratio in participants with ESRD after HD were 90.0% (59.3%, 137%) and 91.3% (64.1%, 130%), respectively. CONCLUSION: Pemigatinib dose should be reduced for patients with severe hepatic or renal impairment, and no dose adjustment is required for patients with moderate hepatic impairment or in ESRD patients undergoing HD.

Evaluation of drug-drug interactions of pemigatinib in healthy participants.

PURPOSE: Pemigatinib (INCB054828), a potent and selective oral fibroblast growth factor receptor 1-3 inhibitor, is a Biopharmaceutical Classification System class II compound with good permeability and pH-dependent solubility that is predominantly metabolized by cytochrome P450 (CYP) 3A. Two drug-drug interaction studies, one with acid-reducing agents, esomeprazole (proton pump inhibitor [PPI]) and ranitidine (histamine-2 [H2] antagonist), and the other with potent CYP3A-modulating agents, itraconazole (CYP3A inhibitor) and rifampin (CYP3A inducer), were performed. METHODS: Both were open-label, fixed-sequence studies conducted in up to 36 healthy participants each, enrolled into two cohorts (n = 18 each). Pemigatinib plasma concentration was measured, and pharmacokinetic parameters were derived by non-compartmental analysis. RESULTS: There was an 88% and 17% increase in pemigatinib area under the plasma drug concentration-time curve (AUC) and maximum plasma drug concentration (Cmax), respectively, with itraconazole, and an 85% and 62% decrease in pemigatinib AUC and Cmax with rifampin coadministration. There was a 35% and 8% decrease in pemigatinib AUC and Cmax, respectively, with esomeprazole, and a 2% decrease in Cmax and 3% increase in AUC with ranitidine coadministration. In both studies, all adverse events reported were grade ≤ 2. CONCLUSION: Coadministration with itraconazole or rifampin resulted in a clinically significant change in pemigatinib exposure. Therefore, coadministration of strong CYP3A inducers with pemigatinib should be avoided, and the dose of pemigatinib should be reduced if coadministration with strong CYP3A inhibitors cannot be avoided. The effect of PPIs/H2 antagonists on pemigatinib exposure was modest, and pemigatinib can be administered without regard to coadministration of PPIs/H2 antagonists.

Antibiotic transformation in an anaerobic membrane bioreactor linked to membrane biofilm microbial activity.

Although extensive research to date has focused on enhancing removal rates of antibiotics from municipal wastewaters, the transformation products formed by anaerobic treatment processes remain understudied. The present work aims to examine the possible roles that the different microbial communities of an anaerobic membrane bioreactor (AnMBR) play in the transformation of antibiotics during wastewater treatment. As part of this work, sulfamethoxazole, erythromycin, and ampicillin were added in separate stages to the influent of the AnMBR at incremental concentrations of 10, 50, and 250 µg/L each. Antibiotic-specific transformation products detected during each stage, as identified by high resolution LC-MS, are reported herein. Results suggest that both isoxazole (sulfamethoxazole) and ß-lactam (ampicillin) ring opening could be facilitated by the AnMBR's bioprocess. Microbial community analysis results indicated that relative activity of the system's suspended biomass consistently shifted towards syntrophic groups throughout the duration of the experiment. Notable differences were also observed between the suspended biomass and the AnMBR's membrane biofilms. Membrane-attached biofilm communities showed high relative activities of several specific methanogenic (Methanothrix and Methanomethylovorans), syntrophic (Syntrophaceae), and sulfate-reducing (Desulfomonile) groups. Such groups have been previously identified as involved in the formation of the antibiotic degradation products observed in the effluent of the AnMBR. The activity of these communities within the biofilms likely confers certain advantages that aid in the biotransformation of the antibiotics tested.

Membrane Fouling Inversely Impacts Intracellular and Extracellular Antibiotic Resistance Gene Abundances in the Effluent of an Anaerobic Membrane Bioreactor.

Anaerobic membrane bioreactors (AnMBRs) can significantly reduce the release of antibiotic resistance elements to the environment. The purpose of this study was to elucidate the role of membrane fouling layers (biofilms) in mitigating the release of intracellular and extracellular antibiotic resistance genes (iARGs and eARGs) from an AnMBR. The AnMBR was equipped with three membrane modules, each exhibiting a different level of fouling. Results showed that the absolute abundance of ARGs decreased gradually in the suspended biomass during operation of the AnMBR. Normalized abundances of targeted ARGs and intI1 were found to be significantly higher in the fouling layers compared to the suspended biomass, implying adsorption or an increased potential for horizontal gene transfer of ARGs in the biofilm. Effluent ARG data revealed that the highly fouled (HF) membrane significantly reduced the absolute abundance of eARGs. However, the HF membrane effluent concomitantly had the highest absolute abundance of iARGs. Nevertheless, total ARG abundance (sum of iARG and eARG) in the effluent of the AnMBR was not impacted by the extent of fouling. These results suggest a need for a combination of different treatment technologies to effectively prevent antibiotic resistance proliferation associated with these two ARG fractions.

Evaluating Antibiotic Resistance Gene Correlations with Antibiotic Exposure Conditions in Anaerobic Membrane Bioreactors.

Anaerobic membrane bioreactors (AnMBRs) are an emerging technology with potential to improve energy efficiency and effluent reuse in mainstream wastewater treatment. However, their contribution to the proliferation of contaminants of emerging concern, such as antibiotic resistance genes (ARGs), remains largely unknown. The purpose of this study was to determine the effect of select influent antibiotics at varying concentrations on the presence and abundance of ARGs in an AnMBR system and its effluent. Quantification of targeted ARGs revealed distinct profiles in biomass and effluent, with genes conferring resistance to different antibiotic classes dominating in biomass (macrolides) and effluent (sulfonamides). Effluent sul1 gene abundance was strongly correlated with abundance of intl1, signifying the potential importance of mobile genetic elements in ARG release from AnMBR systems. The addition of specific antibiotics also affected normalized abundances of their related ARGs, exemplifying the potential impact of selective pressures at both low (10 µg/L) and high (250 µg/L) influent antibiotic concentrations.

Effect of improved contact on reliability of sub-60 nm carbon nanotube vias.

Advances in semiconductor technology due to the aggressive downward scaling of on-chip feature sizes have led to rapid rises in the resistivity and current density of interconnect conductors. As a result, current interconnect materials, Cu and W, are subject to performance and reliability constraints approaching or exceeding their physical limits. Therefore, alternative materials are being actively considered as potential replacements to meet such constraints. The carbon nanotube (CNT) is among the leading replacement candidates for on-chip interconnect vias due to its high aspect-ratio nanostructure and superior current-carrying capacity to Cu and W, as well as other potential candidates. Based on the results for 40 nm and 60 nm top-contact metallized CNT vias, we demonstrate that not only are their current-carrying capacities two orders of magnitude higher than their Cu and W counterparts, they are enhanced by reduced via resistance due to contact engineering facilitated by the first reported contact resistance extraction scheme for a 40 nm linewidth.

Electron-beam and ion-beam-induced deposited tungsten contacts for carbon nanofiber interconnects.

Ion-beam-induced deposition (IBID) and electron-beam-induced deposition (EBID) with tungsten (W) are evaluated for engineering electrical contacts with carbon nanofibers (CNFs). While a different tungsten-containing precursor gas is utilized for each technique, the resulting tungsten deposits result in significant contact resistance reduction. The performance of CNF devices with W contacts is examined and conduction across these contacts is analyzed. IBID-W, while yielding lower contact resistance than EBID-W, can be problematic in the presence of on-chip semiconducting devices, whereas EBID-W provides substantial contact resistance reduction that can be further improved by current stressing. Significant differences between IBID-W and EBID-W are observed at the electrode contact interfaces using high-resolution transmission electron microscopy. These differences are consistent with the observed electrical behaviors of their respective test devices.

Phage phylogeny, molecular signaling, and auxiliary antimicrobial resistance in aerobic and anaerobic membrane bioreactors.

Phage emit communication signals that inform their lytic and lysogenic life cycles. However, little is known regarding the abundance and diversity of the genes associated with phage communication systems in wastewater treatment microbial communities. This study focused on phage communities within two distinct biochemical wastewater environments, specifically aerobic membrane bioreactors (AeMBRs) and anaerobic membrane bioreactors (AnMBRs) exposed to varying antibiotic concentrations. Metagenomic data from the bench-scale systems were analyzed to explore phage phylogeny, life cycles, and genetic capacity for antimicrobial resistance and quorum sensing. Two dominant phage families, Schitoviridae and Peduoviridae, exhibited redox-dependent dynamics. Schitoviridae prevailed in anaerobic conditions, while Peduoviridae dominated in aerobic conditions. Notably, the abundance of lytic and lysogenic proteins varied across conditions, suggesting the coexistence of both life cycles. Furthermore, the presence of antibiotic resistance genes (ARGs) within viral contigs highlighted the potential for phage to transfer ARGs in AeMBRs. Finally, quorum sensing genes in the virome of AeMBRs indicated possible molecular signaling between phage and bacteria. Overall, this study provides insights into the dynamics of viral communities across varied redox conditions in MBRs. These findings shed light on phage life cycles, and auxiliary genetic capacity such as antibiotic resistance and bacterial quorum sensing within wastewater treatment microbial communities.

p38γ mitogen-activated protein kinase (MAPK) confers breast cancer hormone sensitivity by switching estrogen receptor (ER) signaling from classical to nonclassical pathway via stimulating ER phosphorylation and c-Jun transcription.

Estrogen receptor (ER) α promotes breast cancer growth by regulating gene expression through classical estrogen response element (ERE) binding and nonclassical (interaction with c-Jun at AP-1 sites) pathways. ER is the target for anti-estrogens such as tamoxifen (TAM). However, the potential for classical versus nonclassical ER signaling to influence hormone sensitivity is not known. Moreover, anti-estrogens frequently activate several signaling cascades besides the target ER, and the implications of these "off-target" signaling events have not been explored. Here, we report that p38γ MAPK is selectively activated by treatment with TAM. This results in both phosphorylation of ER at Ser-118 and stimulation of c-Jun transcription, thus switching ER signaling from the classical to the nonclassical pathway leading to increased hormone sensitivity. Unexpectedly, phosphorylation at Ser-118 is required for ER to bind both p38γ and c-Jun, thereby promoting ER relocation from ERE to AP-1 promoter sites. Thus, ER/Ser-118 phosphorylation serves as a central mechanism by which p38γ regulates signaling transduction of ER with its inhibitor TAM.

On initial Brain Activity Mapping of episodic and semantic memory code in the hippocampus.

It has been widely recognized that the understanding of the brain code would require large-scale recording and decoding of brain activity patterns. In 2007 with support from Georgia Research Alliance, we have launched the Brain Decoding Project Initiative with the basic idea which is now similarly advocated by BRAIN project or Brain Activity Map proposal. As the planning of the BRAIN project is currently underway, we share our insights and lessons from our efforts in mapping real-time episodic memory traces in the hippocampus of freely behaving mice. We show that appropriate large-scale statistical methods are essential to decipher and measure real-time memory traces and neural dynamics. We also provide an example of how the carefully designed, sometime thinking-outside-the-box, behavioral paradigms can be highly instrumental to the unraveling of memory-coding cell assembly organizing principle in the hippocampus. Our observations to date have led us to conclude that the specific-to-general categorical and combinatorial feature-coding cell assembly mechanism represents an emergent property for enabling the neural networks to generate and organize not only episodic memory, but also semantic knowledge and imagination.

The use of a membrane filtration device to form dried plasma spots for the quantitative determination of guanfacine in whole blood.

RATIONALE: A two-layered polymeric membrane is employed for the formation of separated dried plasma spots from whole blood as an alternative to the direct analysis of whole dried blood spots (DBS). This dried plasma spot (DPS) analysis procedure precludes potential issues of hematocrit differences in whole blood samples while providing pharmokinetic data from plasma rather than whole blood. The described procedure is also semi-automated thus providing a simpler work flow for LC/MS/MS bioanalysis procedures. METHODS: Molecular filtration of red blood cells (RBC) from applied microsamples of whole blood fortified with guanfacine and its stable isotope internal standard was accomplished with a two-layer polymeric membrane substrate. The lower membrane surface containing the separated plasma spot was physically separated from the upper membrane followed by semi-automated direct elution of the sample to an online solid-phase extraction (SPE) cartridge followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). RESULTS: A two-layer membrane sample preparation substrate produced plasma from whole blood without centrifugation which could be directly eluted for semi-automated LC/MS/MS bioanalysis. Standard curves were constructed by plotting peak area ratios between the analyte and the stable isotope labeled internal standard (SIL-IS) versus the nominal concentration in whole blood. A weighted 1/x(2) linear regression was applied to the data from DPS samples. Standard curves were linear over the range 0.25-250 ng/mL human whole blood. The representative regression equation was y = 0.0142x + 0.00248 (R(2) = 0.995) for the described DPS assay. CONCLUSIONS: The described work demonstrates proof-of-principle using membrane sample preparation techniques to form DPS samples from whole blood for subsequent bioanalysis by LC/MS/MS. This approach has the potential to eliminate the hematocrit issues from the current controversy surrounding validation of DBS assays.

Bioanalysis of INCB000928 in human saliva: nonspecific binding and inhomogeneous concentration.

Aim: To develop a bioanalytical method for quantifying INCB000928 in human saliva. Materials & methods: Human centrifuged saliva and human whole saliva were compared for matrix selection. Protein precipitation extraction and HPLC-MS/MS was used for analysis. Results & conclusion: Nonspecific binding of INCB000928 was reduced in whole versus centrifuged saliva. Whole saliva was a preferred matrix for INCB000928 bioanalytical method validation. Incurred sample reanalysis (ISR) using a successfully validated method failed in a healthy volunteer study because of inhomogeneous INCB000928 concentration across sample tube depths. Individual mixing of sample tubes followed by immediate aliquoting corrected the ISR failure, with 97.2% of repeats passing versus 41.7% for the same ISR samples.

Fibrodysplasia ossificans progressiva (FOP) is a very rare condition where bone forms outside the skeleton (ossification), leading to restricted movement, decreased quality of life and shortened life span. Mutations in a gene called ALK2 have been identified as causing FOP. INCB000928 is a novel drug (to be taken by mouth) which inhibits ALK2 activity and prevents ossification in a laboratory mouse model of FOP. Because monitoring of the levels and efficacy of a drug often requires blood draws, which can be taxing in patients with FOP, this study aimed to develop a method to measure INCB000928 levels in saliva. The authors propose a unique procedure to process saliva samples to ensure accurate, reproducible quantitation of INCB000928 levels in saliva.

Bioanalysis of INCB000928 in hemodialysate: prevention of nonspecific binding and validation of surrogate matrices.

Aim: To develop and validate a bioanalytical method for the quantification of INCB000928 in hemodialysate. Materials & methods: Blank dialysate and phosphate-buffered saline were compared with hemodialysate for surrogate matrix selection. Direct addition of internal standard without analyte extraction and a high-performance LC-MS/MS were used for analysis. Results & conclusion: INCB000928 in hemodialysate exhibited strong nonspecific binding to polypropylene containers. In the presence of 10% isopropyl alcohol, the loss of INCB000928 was fully recovered, regardless of pre- or post-addition of the solvent. Blank dialysate and phosphate-buffered saline were determined to be appropriate surrogate matrices by using a three-way cross-comparison and were subsequently validated in the quantitative analysis of INCB000928 in hemodialysate.

Fibrodysplasia ossificans progressiva (FOP) is a very rare disease characterized by congenital malformation of the great toes and progressive heterotopic ossification. The genetic cause of FOP is mutation in the gene ALK2. INCB000928 is a novel and orally available drug that inhibits ALK2 protein activity and has been shown to prevent ossification in a laboratory mouse model of FOP. Patients with end-stage renal disease who undergo hemodialysis may require a different dose of INCB000928. This study showed that INCB000928 was heavily adsorbed by the container wall, resulting in underestimated drug levels in hemodialysate. We present a method to accurately measure INCB000928 levels in hemodialysate by using isopropyl alcohol as an antiadsorption agent and cost-effective surrogate matrix.

Comparison of dietary phosphate absorption after single doses of lanthanum carbonate and sevelamer carbonate in healthy volunteers: a balance study.

BACKGROUND: Lanthanum carbonate and sevelamer carbonate are noncalcium phosphate binders used to treat hyperphosphatemia in patients with chronic kidney disease. This is the first study to compare phosphate absorption from a standardized meal ingested with a typical clinical dose of these binders. STUDY DESIGN: Randomized open-label crossover study. SETTINGS & PARTICIPANTS: Healthy volunteers were confined to a clinical research center during 4 study periods. Of 31 volunteers randomly assigned, 19 completed all treatments and 18 were analyzed in the pharmacodynamic set (1 was excluded because of vomiting). INTERVENTION: Participants were assigned in random order to meal alone, meal plus lanthanum carbonate (1 tablet containing 1,000 mg of elemental lanthanum), and meal plus sevelamer carbonate (three 800-mg tablets). The gastrointestinal tract was cleared, the meal was ingested (± treatment), and rectal effluent was collected. In a fourth period, volunteers repeated the study procedures while fasting. OUTCOMES: The primary end point, net phosphate absorption, was analyzed using a mixed-effect linear model. MEASUREMENTS: Phosphorus content of effluent and duplicate meal samples were measured using inductively coupled plasma-optical emission spectroscopy. RESULTS: The standard meal contained ∼375 mg of phosphate, 75% of which was absorbed (net absorption, 281.7 ± 14.1 mg [adjusted mean ± standard error]). Lanthanum carbonate decreased net phosphate absorption by 45% (net absorption, 156.0 ± 14.2 mg) compared with 21% (net absorption, 221.8 ± 14.1 mg) for sevelamer carbonate (P < 0.001). Lanthanum carbonate bound 135.1 ± 12.3 mg of phosphate, whereas sevelamer carbonate bound 63.2 ± 12.3 mg, a 71.9-mg difference (95% CI, 40.0-103.8; P < 0.001). Per tablet, this equates to 135 mg of phosphate bound with lanthanum carbonate versus 21 mg with sevelamer carbonate. LIMITATIONS: A single-dose study. CONCLUSIONS: In healthy volunteers, 1,000 mg of lanthanum carbonate decreased phosphate absorption by 45% compared with a 21% decrease with 2,400 mg of sevelamer carbonate.

Investigation of Fats, Oils, and Grease Co-digestion With Food Waste in Anaerobic Membrane Bioreactors and the Associated Microbial Community Using MinION Sequencing.

Co-digestion of fats, oils, and grease (FOG) with food waste (FW) can improve the energy recovery in anaerobic membrane bioreactors (AnMBRs). Here, we investigated the effect of co-digestion of FW and FOG in AnMBRs at fat mass loading of 0.5, 0.75, and 1.0 kg m-3 day-1 with a constant organic loading rate of 5.0 gCOD L-1 day-1 in both a single-phase (SP) and two-phase (TP) configuration. A separate mono-digestion of FW at an identical organic loading rate was used as the benchmark. During co-digestion, higher daily biogas production, ranging from 4.0 to 12.0%, was observed in the two-phase methane phase (TP-MP) reactor compared to the SP reactor, but the difference was statistically insignificant (p > 0.05) due to the high variability in daily biogas production. However, the co-digestion of FW with FOG at 1.0 kg m-3 day-1 fat loading rate significantly (p < 0.05) improved daily biogas production in both the SP (11.0%) and TP (13.0%) reactors compared to the mono-digestion of FW. Microbial community analyses using cDNA-based MinION sequencing of weekly biomass samples from the AnMBRs revealed the prevalence of Lactobacillus (92.2-95.7% relative activity) and Anaerolineaceae (13.3-57.5% relative activity), which are known as fermenters and fatty acid degraders. Syntrophic fatty acid oxidizers were mostly present in the SP and TP-MP reactors, possibly because of the low pH and short solid retention time (SRT) in the acid phase digesters. A greater abundance of the mcrA gene copies (and methanogens) was observed in the SP and MP reactors compared to the acid-phase (AP) reactors. This study demonstrates that FW and FOG can be effectively co-digested in AnMBRs and is expected to inform full-scale decisions on the optimum fat loading rate.

In vivo evidence for NMDA receptor-mediated excitotoxicity in a murine genetic model of Huntington disease.

N-methyl-D-aspartate receptor (NMDAR)-mediated excitotoxicity is implicated as a proximate cause of neurodegeneration in Huntington Disease (HD). This hypothesis has not been tested rigorously in vivo. NMDAR-NR2B subunits are a major NR2 subunit expressed by striatal medium spiny neurons that degenerate in HD. To test the excitotoxic hypothesis, we crossed a well validated murine genetic model of HD (Hdh((CAG)150)) with a transgenic line overexpressing NMDAR-NR2B subunits. In the resulting double-mutant line, we show exacerbation of selective striatal neuron degeneration. This is the first direct in vivo evidence of NR2B-NMDAR-mediated excitotoxicity in the context of HD. Our results are consistent with previous suggestions that direct and/or indirect interactions of mutant huntingtin with NMDARs are a proximate cause of neurodegeneration in HD.

Towards transgenic primates: What can we learn from mouse genetics?

Considering the great physiological and behavioral similarities with humans, monkeys represent the ideal models not only for the study of complex cognitive behavior but also for the preclinical research and development of novel therapeutics for treating human diseases. Various powerful genetic technologies initially developed for making mouse models are being explored for generating transgenic primate models. We review the latest genetic engineering technologies and discuss the potentials and limitations for systematic production of transgenic primates.

Effect of Itraconazole or Rifampin on Itacitinib Pharmacokinetics When Administered Orally in Healthy Subjects.

Itacitinib is a potent, selective JAK-1 inhibitor currently in phase 3 development for the treatment of acute and chronic graft-versus-host disease (GVHD) in combination with corticosteroids. Itacitinib is primarily eliminated via metabolism by cytochrome P-450 (CYP)3A4 with minimal renal elimination. A drug-drug interaction study was conducted to evaluate the impact of the strong CYP3A inhibitor itraconazole or the strong CYP3A4 inducer rifampin on the pharmacokinetics of itacitinib in healthy volunteers. In cohort 1, subjects received 200 mg sustained release (SR) tablets of itacitinib on days 1 and 6 and 200 mg itraconazole on days 2-7. In cohort 2, subjects received 200 mg SR itacitinib on days 1 and 9 and 600 mg rifampin on days 2-9. Thirty-six subjects were enrolled, 18 in each cohort with 17 completing itacitinib dosing in cohort 1 and 15 completing itacitinib dosing in cohort 2. Coadministration of itraconazole with itacitinib resulted in a nearly 5-fold increase in area under the concentration-time curve (AUC0-∞ ) (geometric mean ratio [GMR] 4.88, 90%Cl 4.17-5.72) and an ∼3-fold increase in peak concentration (Cmax ) (GMR 3.15, 90%Cl 2.58-3.54). Coadministration of rifampin with itacitinib resulted in a nearly 80% decrease in AUC0-∞ (GMR 0.208, 90%Cl 0.173, 0.249) and Cmax (GMR 0.231, 90%Cl 0.195, 0.274). Results of this study informed the study design of the phase 3 GVHD protocols with regard to coadministration of strong CYP3A inhibitors and CYP3A4 inducers. These data combined with phase 3 data will inform final dosing recommendations.