Estimation of FMO3 Ontogeny by Mechanistic Population Pharmacokinetic Modelling of Risdiplam and Its Impact on Drug-Drug Interactions in Children.

BACKGROUND AND OBJECTIVE: Spinal muscular atrophy (SMA) is a progressive neuromuscular disease caused by insufficient levels of survival motor neuron (SMN) protein. Risdiplam (EvrysdiTM) increases SMN protein and is approved for the treatment of SMA. Risdiplam has high oral bioavailability and is primarily eliminated through hepatic metabolism by flavin-containing monooxygenase3 (FMO3) and cytochrome P450 (CYP) 3A, by 75% and 20%, respectively. While the FMO3 ontogeny is critical input data for the prediction of risdiplam pharmacokinetics (PK) in children, it was mostly studied in vitro, and robust in vivo FMO3 ontogeny is currently lacking. We derived in vivo FMO3 ontogeny by mechanistic population PK modelling of risdiplam and investigated its impact on drug-drug interactions in children. METHODS: Population and physiologically based PK (PPK and PBPK) modelling conducted during the development of risdiplam were integrated into a mechanistic PPK (Mech-PPK) model to estimate in vivo FMO3 ontogeny. A total of 10,205 risdiplam plasma concentration-time data from 525 subjects aged 2 months-61 years were included. Six different structural models were examined to describe the in vivo FMO3 ontogeny. Impact of the newly estimated FMO3 ontogeny on predictions of drug-drug interaction (DDI) in children was investigated by simulations for dual CYP3A-FMO3 substrates including risdiplam and theoretical substrates covering a range of metabolic fractions (fm) of CYP3A and FMO3 (fmCYP3A:fmFMO3 = 10%:90%, 50%:50%, 90%:10%). RESULTS: All six models consistently predicted higher FMO3 expression/activity in children, reaching a maximum at the age of 2 years with an approximately threefold difference compared with adults. Different trajectories of FMO3 ontogeny in infants < 4 months of age were predicted by the six models, likely due to limited observations for this age range. Use of this  in vivo FMO3 ontogeny function improved prediction of risdiplam PK in children compared to in vitro FMO3 ontogeny functions. The simulations of theoretical dual CYP3A-FMO3 substrates predicted comparable or decreased CYP3A-victim DDI propensity in children compared to adults across the range of fm values. Refinement of FMO3 ontogeny in the risdiplam model had no impact on the previously predicted low CYP3A-victim or -perpetrator DDI risk of risdiplam in children. CONCLUSION: Mech-PPK modelling successfully estimated in vivo FMO3 ontogeny from risdiplam data collected from 525 subjects aged 2 months-61 years. To our knowledge, this is the first investigation of in vivo FMO3 ontogeny by population approach using comprehensive data covering a wide age range. Derivation of a robust in vivo FMO3 ontogeny function has significant implications on the prospective prediction of PK and DDI in children for other FMO3 substrates in the future, as illustrated in the current study for FMO3 and/or dual CYP3A-FMO3 substrates. CLINICAL TRIAL REGISTRY NUMBERS: NCT02633709, NCT03032172, NCT02908685, NCT02913482, NCT03988907.

Artificial Intelligence Competencies in Postgraduate Medical Training in Germany.

Routine medical care is to be transformed by the introduction of artificial intelligence (AI), requiring medical professionals to acquire a novel set of skills. We assessed the density of AI learning objectives and the availability of courses containing AI content in postgraduate medical education in Germany. The results reveal general paucity in AI learning objectives and content across (sub-)specialty training and continuing medical education (CME) in Germany. Innovative and regulatory solutions are needed to herald an era of physicians competent in navigating medical AI applications.

Disease Modeling and Model-Based Meta-Analyses to Define a New Direction for a Phase III Program of Gantenerumab in Alzheimer's Disease.

Selecting the right dose is a significant challenge in designing clinical development programs, especially for slowly progressing diseases lacking predictive biomarkers of efficacy that may require long-term treatment to assess clinical benefit. Gantenerumab, a fully human monoclonal antibody (mAb) that binds to aggregated amyloid-beta, was tested in two 24-month phase III studies (NCT01224106, NCT02051608) in participants with prodromal and mild Alzheimer's disease (AD), respectively. Dosing in the first phase III study was suspended after a preplanned interim futility analysis in 2014. Subsequently, a dose-response relationship was observed in a subgroup of fast AD progressors that, together with contemporary aducanumab (another anti-amyloid-beta mAb) data, indicated higher doses may be needed for clinical efficacy. The gantenerumab phase III studies were therefore transformed into dose-finding, open-label extension (OLE) trials. Two exposure-response models were developed to support dose selection via simulations for the OLEs: a pharmacokinetics (PK)/PET (positron emission tomography) model describing amyloid removal using PET data from low-dose gantenerumab and high-dose aducanumab, and a PK/ARIA-E (amyloid-related imaging abnormalities-edema) model describing the occurrence of ARIA-E events leveraging an existing bapineuzumab model. Multiple regimens were designed to gradually up-titrate participants to the target dose of 1,200 mg gantenerumab every 4 weeks to mitigate the increased risk of ARIA-E events that may be associated with higher doses of anti-amyloid-beta antibodies. Favorable OLE data that matched well with model predictions supported the decision to continue the gantenerumab clinical development program and further apply model-based analytical techniques to optimize the design of new phase III studies.

Population pharmacokinetic analysis of entrectinib in pediatric and adult patients with advanced/metastatic solid tumors: support of new drug application submission.

PURPOSE: Entrectinib (ROZLYTREK®) is a CNS-active, potent, and selective inhibitor of ROS1, TRK A/B/C, and ALK kinase activity. It was recently approved for the treatment of ROS1-positive non-small cell lung cancer and NTRK gene fusion-positive solid tumors. The main objective of this analysis was to characterize the pharmacokinetics (PK) of entrectinib and its main active metabolite, M5. METHODS: A total of 276 cancer patients receiving oral entrectinib were included in the analysis. A model-based population approach was used to characterize the PK profiles of both entities using NONMEM® 7.4. A joint model captures the PK of both entrectinib and M5. The effects of pH modifiers, formulation, weight, age, and sex on model parameters were assessed. Model performance was evaluated using visual predictive checks (VPCs). RESULTS: The absorption of entrectinib was best described using a sequential zero- and first-order absorption model and the disposition with one-compartment model for each entity with linear elimination. Moderate-to-high between-patient variability was estimated in model parameters (from 30.8% for the apparent clearance of entrectinib to 122% for the first-order absorption rate constant). Theory-based allometric scaling using body weight on clearances and volumes and a 28% lower relative bioavailability of the F1 formulation in pediatric patients were retained in the model. The VPC confirmed the good predictive performance of the PopPK model. CONCLUSIONS: A robust population PK model was built and qualified for entrectinib and M5, describing linear PK for both entities. This model was used to support the ROZLYTREK® new drug application.

Pharmacometric analyses of alectinib to facilitate approval of the optimal dose for the first-line treatment of anaplastic lymphoma kinase-positive non-small cell lung cancer.

Alectinib is an anaplastic lymphoma kinase (ALK) inhibitor approved for treatment of ALK-positive non-small cell lung cancer. Population pharmacokinetic (PK) models were developed for alectinib and its major active metabolite M4 using phase I/II PK data in crizotinib-failed patients (N = 138). The PK profiles were best described by two separate models with similar structure for both entities: open one-compartment models with sequential zero/first-order input and first-order elimination rate. Body weight with fixed allometric scaling factor on clearance and volume of both entities was the only significant covariate. Bayesian feedback analyses of the PK data collected from Japanese and global treatment-naïve patients in phase III studies (N = 334) confirmed the body weight effect. Landmark Cox proportional hazards analyses of progression-free survival in treatment-naïve patients identified the average molar concentrations of both entities alectinib and M4 during the first 6 weeks of treatment as a significant covariate, with an optimal response achieved for concentrations above 1040 nmol/L. With 600 mg twice daily (b.i.d.), 92% of global patients are above this threshold concentration, compared with only 43% of patients with 300 mg b.i.d. In Japan, where the body weight distribution is lower, the approved 300 mg b.i.d. dose brings about 70% of Japanese patients above this threshold. Logistic regression analyses found no significant relationship between the combined alectinib-M4 molar concentration and first occurrence of adverse events. These pharmacometric results were used to expedite and facilitate regulatory approvals of 600 mg b.i.d. for first-line ALK-positive NSCLC in the United States and European Union in 2017 and in China in 2018.

Physiologically-Based Pharmacokinetic Modelling of Entrectinib Parent and Active Metabolite to Support Regulatory Decision-Making.

BACKGROUND AND OBJECTIVE: Entrectinib is a selective inhibitor of ROS1/TRK/ALK kinases, recently approved for oncology indications. Entrectinib is predominantly cleared by cytochrome P450 (CYP) 3A4, and modulation of CYP3A enzyme activity profoundly alters the pharmacokinetics of both entrectinib and its active metabolite M5. We describe development of a combined physiologically based pharmacokinetic (PBPK) model for entrectinib and M5 to support dosing recommendations when entrectinib is co-administered with CYP3A4 inhibitors or inducers. METHODS: A PBPK model was established in Simcyp® Simulator. The initial model based on in vitro-in vivo extrapolation was refined using sensitivity analysis and non-linear mixed effects modeling to optimize parameter estimates and to improve model fit to data from a clinical drug-drug interaction study with the strong CYP3A4 inhibitor, itraconazole. The model was subsequently qualified against clinical data, and the final qualified model used to simulate the effects of moderate to strong CYP3A4 inhibitors and inducers on entrectinib and M5 pharmacokinetics. RESULTS: The final model showed good predictive performance for entrectinib and M5, meeting commonly used predictive performance acceptance criteria in each case. The model predicted that co-administration of various moderate CYP3A4 inhibitors (verapamil, erythromycin, clarithromycin, fluconazole, and diltiazem) would result in an average increase in entrectinib exposure between 2.2- and 3.1-fold, with corresponding average increases for M5 of approximately 2-fold. Co-administration of moderate CYP3A4 inducers (efavirenz, carbamazepine, phenytoin) was predicted to result in an average decrease in entrectinib exposure between 45 and 79%, with corresponding average decreases for M5 of approximately 50%. CONCLUSIONS: The model simulations were used to derive dosing recommendations for co-administering entrectinib with CYP3A4 inhibitors or inducers. PBPK modeling has been used in lieu of clinical studies to enable regulatory decision-making.

Population pharmacokinetic and exposure-response analyses of intravenous and subcutaneous rituximab in patients with chronic lymphocytic leukemia.

A subcutaneous formulation of the anti-CD20 antibody rituximab has been developed. Fixed-dose subcutaneous rituximab delivers noninferior serum trough concentrations (Ctrough ), ensuring similar target saturation and comparable efficacy/safety, to intravenous rituximab, but with simplified and shortened preparation and administration. We aimed to characterize the pharmacokinetic (PK) and exposure-response properties of subcutaneous rituximab. Data from two clinical trials were analyzed to describe PKs and pharmacodynamics in patients with chronic lymphocytic leukemia following intravenous and subcutaneous rituximab administration. Intravenous and subcutaneous rituximab were described by a linear two-compartment population PK model with time-dependent and time-independent clearances, and first-order subcutaneous absorption. Main covariates influencing exposure were body size and baseline white blood cell count. Occurrence of adverse events was not correlated with rituximab exposure. Although greater and more sustainable B-cell depletion was observed with higher exposure, inherent limitations to the data (use of one dose level, and time-dependent and target-impacted PKs) prevented reliable assessment of exposure-response relationships.

A Baseline Score to Predict Response to Ranibizumab Treatment in Neovascular Age-Related Macular Degeneration.

Purpose: What are the patient characteristics predictive of response to ranibizumab treatment? Methods: Model-based characterization of best-corrected visual acuity (BCVA) time profiles of patients with neovascular age-related macular degeneration under ranibizumab or sham treatment based on 24-month observations of BCVA in 2419 patients from randomized multicenter phase 3 trials of ranibizumab: ANCHOR, MARINA, PIER, and HARBOR. Goodness-of-fit plots and precision of parameter estimates were used for measure of accuracy. Results: The model incorporates a long-term effect on disease progression and an additive and more potent short-term effect of ranibizumab. Response to ranibizumab treatment and progression of the disease were found to be a function of seven baseline characteristics (visual acuity, age, leakage size, central retinal lesion thickness, presence or absence of cyst, type of choroidal neovascularization (CNV), and size of pigment epithelium detachment). A composite score of these seven baseline characteristics was derived and used to categorize response to ranibizumab treatment. The ranibizumab treatment arms of two proof-of-concept studies held out from the model development were used to validate the methodology. Conclusions: A composite score based on seven patient characteristics prior to treatment could be used to discriminate patients with predicted insufficient response to anti-vascular endothelial growth factor treatment. Translational Relevance: The method could be used to create a virtual ranibizumab treatment arm in clinical trials or to reduce the size of a ranibizumab active control arm.

Quantitative Clinical Pharmacology Supports the Bridging From i.v. Dosing and Approval of s.c. Rituximab in B-Cell Hematological Malignancies.

A fixed-dose subcutaneous (s.c.) formulation of the anti-CD20 antibody, rituximab, has been developed to address safety, infusion time, and patient comfort concerns relating to intravenous (i.v.) dosing, and has been approved based upon a pharmacokinetic (PK)-clinical bridging strategy, which demonstrated noninferiority of s.c. vs. i.v. dosing in malignancies, including follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL). A clinical development plan was undertaken to identify rituximab s.c. doses achieving noninferior exposure to rituximab i.v., and to confirm PK-clinical bridging, with the same efficacy and similar safety. This drew upon data from 1,579 patients with FL, CLL, or diffuse large B-cell lymphoma in 5 clinical studies, and showed minimum steady-state serum concentration (Ctrough ) as the most appropriate exposure bridging measure. Population PK models were developed, simulations were run using covariates and PK parameters from clinical studies, and exposure-efficacy and -safety analyses performed. Population PKs showed a two-compartment model with time-dependent and -independent clearances. Clearance and volume were predominantly influenced by body surface area; disposition and elimination were similar for the s.c. and i.v. formulations. After s.c. administration, patients with FL and CLL achieved noninferior exposures to i.v. dosing. Overall, rituximab exposure and route of administration did not influence clinical responses in patients with FL or CLL, and there was no association between exposure and safety events. Ctrough was shown to be an effective pharmacologic-clinical bridging parameter for rituximab in patients with FL or CLL. Clinically effective exposures are achieved with either s.c. or i.v. dosing.

Exposure-Bleeding Count Modeling of Emicizumab for the Prophylaxis of Bleeding in Persons with Hemophilia A with/Without Inhibitors Against Factor VIII.

BACKGROUND AND OBJECTIVE: Emicizumab is a monoclonal antibody that bridges activated coagulation factor IX and factor X to restore effective hemostasis in persons with hemophilia A. It is indicated for routine prophylaxis of bleeding episodes in persons with hemophilia A. The aim of the present study is to describe the exposure-response relationship between emicizumab concentrations and bleeding frequency, and to confirm adequate bleeding control of the investigated dosing regimens 1.5 mg/kg once weekly, 3 mg/kg every 2 weeks, and 6 mg/kg every 4 weeks. METHODS: Treated bleeding events were pooled from 445 persons with hemophilia A with and without inhibitors against factor VIII, participating in six clinical studies. Emicizumab concentrations were predicted using a previously developed population pharmacokinetic model. A count model was used to quantify the exposure-response relationship. These models were used to illustrate the relationship between emicizumab concentrations and cumulative count of bleeding over 1 year (annualized bleeding rate). RESULTS: The final exposure-response model, based on a generalized Poisson distribution and an inhibitory Emax relationship, adequately describes the relationship between daily emicizumab concentrations and daily bleed frequency. A significant effect of factor VIII prophylaxis among persons with hemophilia A without inhibitors was found. Annualized bleeding rate simulations show that the three emicizumab dosing regimens maintain the concentrations close to the plateau of the effect. At the average steady-state concentration across all regimens (53.5 µg/mL), the predicted mean annualized bleeding rate is 1.28, corresponding to a 94.0% reduction from baseline. CONCLUSIONS: These results confirm that average emicizumab concentrations achieved with all three emicizumab dosing regimens provide adequate bleeding control.

cLRT-Mod: An efficient methodology for pharmacometric model-based analysis of longitudinal phase II dose finding studies under model uncertainty.

Within the challenging context of phase II dose-finding trials, longitudinal analyses may increase drug effect detection power compared to an end-of-treatment analysis. This work proposes cLRT-Mod, a pharmacometric adaptation of the MCP-Mod methodology, which allows the use of nonlinear mixed effect models to first detect a dose-response signal and then identify the doses for the confirmatory phase while accounting for model structure uncertainty. The method was evaluated through extensive clinical trial simulations of a hypothetical phase II dose-finding trial using different scenarios and comparing different methods such as MCP-Mod. The results show an increase in power using cLRT with longitudinal data compared to an EOT multiple contrast tests for scenarios with small sample size and weak drug effect while maintaining pre-specifiability of the models prior to data analysis and the nominal type I error. This work shows how model averaging provides better coverage probability of the drug effect in the prediction step, and avoids under-estimation of the size of the confidence interval. Finally, for illustration purpose cLRT-Mod was applied to the analysis of a real phase II dose-finding trial.

Population pharmacokinetic analysis of RO5459072, a low water-soluble drug exhibiting complex food-drug interactions.

AIMS: RO5459072, a cathepsin-S inhibitor, Biopharmaceutics Classification System class 2 and P-glycoprotein substrate, exhibited complex, nonlinear pharmacokinetics (PK) while fasted that seemed to impact both the absorption and the disposition phases. When given with food, all nonlinearities disappeared. Physiologically based PK (PBPK) modelling attributed those nonlinearities to dose-dependent solubilisation and colonic absorption. The objective of this population PK analysis was to complement the PBPK analysis. METHODS: PK profiles in 39 healthy volunteers after first oral dosing (1-600 mg) while fasted or fed in single and multiple ascending dose studies were analysed using population compartmental modelling. RESULTS: The PK of RO5459072 while fed was characterized by a 1-compartmental PK model with linear absorption and elimination. The nonlinearities while fasted were captured using dose dependent bioavailability and 2 sequential first-order absorption phases: one following drug administration and one occurring 11 hours later and only for doses >10 mg. The bioavailability in the first absorption phase increased between 1 and 10 mg and then decreased with dose, in agreement with in vitro dissolution and solubility studies. The remaining fraction of doses to be absorbed by the second absorption phase was found to have a bioavailability similar to that in the first absorption phase. CONCLUSION: The population PK model supported that dissolution- and solubility-limited absorption from the proximal and distal intestine alone explains the nonlinear PK of RO5459072 in fasted state and the linear PK in fed state. This work, together with the PBPK analysis, raised our confidence in the understanding of this complex PK.

Phytohormone production by the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal symbiosis is a mutualistic interaction between most land plants and fungi of the glomeromycotina subphylum. The initiation, development and regulation of this symbiosis involve numerous signalling events between and within the symbiotic partners. Among other signals, phytohormones are known to play important roles at various stages of the interaction. During presymbiotic steps, plant roots exude strigolactones which stimulate fungal spore germination and hyphal branching, and promote the initiation of symbiosis. At later stages, different plant hormone classes can act as positive or negative regulators of the interaction. Although the fungus is known to reciprocally emit regulatory signals, its potential contribution to the phytohormonal pool has received little attention, and has so far only been addressed by indirect assays. In this study, using mass spectrometry, we analyzed phytohormones released into the medium by germinated spores of the arbuscular mycorrhizal fungus Rhizophagus irregularis. We detected the presence of a cytokinin (isopentenyl adenosine) and an auxin (indole-acetic acid). In addition, we identified a gibberellin (gibberellin A4) in spore extracts. We also used gas chromatography to show that R. irregularis produces ethylene from methionine and the α-keto γ-methylthio butyric acid pathway. These results highlight the possibility for AM fungi to use phytohormones to interact with their host plants, or to regulate their own development.

Population Pharmacokinetic Analysis and Exploratory Exposure-Bleeding Rate Relationship of Emicizumab in Adult and Pediatric Persons with Hemophilia A.

BACKGROUND: Emicizumab is a bispecific monoclonal antibody developed for routine prophylaxis of bleeding in people with hemophilia A (PwHA). This work characterizes the pharmacokinetics of emicizumab in adult and pediatric PwHA, identifies factors contributing to its between-person variabilities, compares the pharmacokinetics following different dosing regimens, and makes a descriptive assessment of the exposure-bleeding events relationship. METHODS: A population pharmacokinetic model was developed, using a database of 389 PwHA from five clinical studies. Potential baseline covariate effects were assessed, including body size, age, race, presence of factor VIII inhibitors, and albumin levels. Using the population pharmacokinetic model, the estimated individual average exposures over the administration period were compared across categories of annualized bleeding rate. RESULTS: A linear one-compartment model with first-order absorption and elimination processes and no lag time best described the emicizumab pharmacokinetics. Body weight, albumin levels, age, and black race were statistically correlated with primary pharmacokinetic parameters, but only body weight had an important influence on exposure. Dosing regimens of 1.5 mg/kg weekly, 3 mg/kg every 2 weeks, or 6 mg/kg every 4 weeks provided similar average concentrations at steady state. A trend for lower exposure was observed in the small proportion of PwHA having an annualized bleeding rate > 4 (11.9%), suggesting that reducing exposure to lower levels may potentially increase the bleeding risk. CONCLUSIONS: Emicizumab pharmacokinetics in PwHA was described with dose-independent parameters. Body weight was an important predictor of emicizumab pharmacokinetics. All three dosing regimens are predicted to achieve similar exposure associated with clinically meaningful prevention of bleeding.

Model-based approach for methoxy polyethylene glycol-epoetin beta drug development in paediatric patients with anaemia of chronic kidney disease.

AIMS: Methoxy polyethylene glycol-epoetin beta (continuous erythropoietin receptor activator, C.E.R.A.) is used for the treatment of anaemia in adults with chronic kidney disease (CKD). Patients treated with shorter-acting erythropoiesis-stimulating agents up to three times weekly can be switched to once-monthly C.E.R.A.. Doses can be adjusted on a monthly basis based on haemoglobin (Hb) levels during the preceding period. A model-based approach was applied to optimise C.E.R.A. development, more specifically the confirmatory trial of the paediatric plan. METHODS: Pharmacokinetic and pharmacodynamic data from a phase II paediatric study and phase II and III adult studies were analysed together using modelling and simulation to determine the pharmacokinetic/pharmacodynamic characteristics of C.E.R.A. in a broad population. Model-based simulations of C.E.R.A. treatment outcomes in paediatric patients were performed, notably when administered subcutaneously and compared to clinical and real-world data. RESULTS: Age and body weight explained differences in pharmacokinetics, while the pharmacodynamic characteristics of C.E.R.A. were similar between adult and paediatric populations. Simulated Hb levels (mean and 95% prediction interval 10.9 [10.6, 11.2] g dL-1 ) and C.E.R.A. doses (median and 95% prediction interval 105 [72, 159] µg) 20 weeks after switching to subcutaneous C.E.R.A. were confirmed by observed real-world data from International Pediatric Dialysis Network registries (mean Hb was 10.8 g dL-1 and median C.E.R.A. dose was 100 µg). CONCLUSIONS: These analyses have facilitated optimisation of the C.E.R.A. development programme in paediatric patients with anaemia of CKD to provide this patient population with faster access to the drug while avoiding unnecessary clinical trial exposure and related monitoring burden in children.

Laccaria bicolor MiSSP8 is a small-secreted protein decisive for the establishment of the ectomycorrhizal symbiosis.

The ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long-term and intimate relationship between the soil-borne fungi and the roots of trees. Mycorrhiza-induced Small-Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8-RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to the lack of hyphal aggregation. MiSSP8 C-terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small-secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation.