Population Pharmacokinetics of Remimazolam in Procedural Sedation With Nonhomogeneously Mixed Arterial and Venous Concentrations.

Remimazolam is an ultra-short acting benzodiazepine under development for procedural sedation and general anesthesia. Population pharmacokinetic analysis (PopPK) was conducted for remimazolam with arterial and venous samples previously, but results were limited by arterial-venous concentration differences and inaccurate central volume of distribution (V1) estimates. A new model was developed to describe covariate effects after accounting for arterial-venous differences. Arterial and venous plasma concentration-time data from 11 clinical trials were pooled for PopPK. Data from two constant-rate infusion studies were used to account for venous-to-arterial (VtoA) ratio within residual error and to accurately estimate V1. V1 and VtoA ratio from the pilot model were applied to the full dataset, where the optimal fixed/random effects and covariates were assessed. VtoA ratio was described using a maximum effect (Emax ) model during infusion and as a constant postdose. V1 was estimated as 4.83 L for a 70 kg subject and interindividual variability (IIV) on V1 could only be estimated in studies with early concentrations. IIV on clearance was low (22.9%). Covariates included effects of sex on clearance (women 10% > men), and race on clearance and steady-state volume of distribution (African Americans 16% < other races). Arterial-venous concentration differences were best described using an Emax model during infusion with a constant ratio after infusion, resulting in low residual error (20.7%). There are no clinically relevant dose adjustments needed for any covariates based on pharmacokinetic differences.

Population pharmacokinetic/pharmacodynamic modeling for remimazolam in the induction and maintenance of general anesthesia in healthy subjects and in surgical subjects.

STUDY OBJECTIVE: To evaluate factors affecting variability in response to remimazolam in general anesthesia. DESIGN: Plasma concentration-time data from 11 Phase 1-3 clinical trials were pooled for the population pharmacokinetic (popPK) analysis and concentration-bispectral index (BIS) data were pooled from 8 trials for popPK-PD analysis. A 3-compartment model with allometric exponents on clearance and volume described remimazolam concentrations over time. An effect compartment model with an inhibitory sigmoid Emax model was fit to the concentration-BIS data. Simulations were performed to assess sedation in general anesthesia and post-surgical sedation in healthy and sensitive populations. SETTING: General anesthesia and post-surgical sedation. PATIENTS: 689 subjects included in popPK and 604 subjects included in popPK-PD. Most subjects (>85%) were ASA Class 1 or 2, with the remaining subjects being ASA Class 3. INTERVENTIONS: Serial plasma concentrations and BIS scores. MEASUREMENTS: Standard intra-operative monitoring. MAIN RESULTS: PopPK model included an effect of extracorporeal circulation, ASA class, and sex on PK and a time-dependent clearance (~30% lower at 24 h) that was not related to cumulative dose. Co-administered remifentanil had a synergistic decrease in BIS with remimazolam. Remimazolam IC50 increased with cumulative dose. Onset was faster in overweight subjects and slower in Asian subjects. If using a weight-based regimen, simulations showed that remimazolam 6 mg/kg/h until loss of consciousness followed by 1 mg/kg/h during general anesthesia and 0.25 mg/kg/h for post-surgical sedation for up to 24 h is optimal, regardless of ASA class or sensitivity of subjects. CONCLUSIONS: If using a weight-based regimen, results illustrated an appropriate regimen of remimazolam for general anesthesia and post-surgical sedation in general and sensitive populations, although lower doses can be considered in elderly patients with a significant disease burden or in ASA Class 3 patients. The time-dependent change in clearance is not clinically relevant for up to 24 h.

Time-to-Event Modeling for Remimazolam for the Indication of Induction and Maintenance of General Anesthesia.

Remimazolam is an ultra-short-acting benzodiazepine being investigated for induction and maintenance of general anesthesia and for procedural sedation. This dose-response analysis of 4 phase 2-3 studies evaluated covariates that may impact the pharmacodynamic profile (based on theoretical pharmacokinetic principles) and require dose adjustments in subpopulations, particularly elderly, and if remimazolam has cumulative properties. Covariates affecting the time to loss of consciousness and time to extubation were evaluated using Cox proportional hazards models. Factors affecting steady-state infusion rate required to produce adequate sedation were evaluated using linear regression. Variability in time to loss of consciousness was explained by induction dose, age, body mass index, and time from initiation of opioids to initiation of remimazolam. The steady-state infusion rate producing adequate sedation was higher in European than Japanese subjects due to differences in study design. American Society of Anesthesiologists physical status class 3 subjects had a 28% lower maintenance infusion rate than class 1 subjects. Other statistically significant covariates (American Society of Anesthesiologists class 2, estimated glomerular filtration rate, and sex) resulted in small (≤14%), non-clinically relevant differences. Factors affecting time to extubation included the last infusion rate (ie, tapering), the bispectral index score at the end of infusion, and sex. The time to extubation after remimazolam did not increase with increased cumulative dose of remimazolam or duration of surgery. This evaluation of remimazolam's pharmacodynamic profile, in the absence of pharmacokinetic data, informed dosing recommendations and showed that remimazolam does not have cumulative properties in the general anesthesia setting.

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression.

Despite critical roles in development and cancer, the mechanisms that specify invasive cellular behavior are poorly understood. Through a screen of transcription factors in Caenorhabditis elegans, we identified G1 cell-cycle arrest as a precisely regulated requirement of the anchor cell (AC) invasion program. We show that the nuclear receptor nhr-67/tlx directs the AC into G1 arrest in part through regulation of the cyclin-dependent kinase inhibitor cki-1. Loss of nhr-67 resulted in non-invasive, mitotic ACs that failed to express matrix metalloproteinases or actin regulators and lack invadopodia, F-actin-rich membrane protrusions that facilitate invasion. We further show that G1 arrest is necessary for the histone deacetylase HDA-1, a key regulator of differentiation, to promote pro-invasive gene expression and invadopodia formation. Together, these results suggest that invasive cell fate requires G1 arrest and that strategies targeting both G1-arrested and actively cycling cells may be needed to halt metastatic cancer.

Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins.

The capability to conditionally inactivate gene function is essential for understanding the molecular basis of development. In gene and mRNA targeting approaches, protein products can perdure, complicating genetic analysis. Current methods for selective protein degradation require drug treatment or take hours for protein removal, limiting their utility in studying rapid developmental processes in vivo. Here, we repurpose an endogenous protein degradation system to rapidly remove targeted C. elegans proteins. We show that upon expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, proteins tagged with the ZF1 zinc-finger domain can be quickly degraded in all somatic cell types examined with temporal and spatial control. We demonstrate that genes can be engineered to become conditional loss-of-function alleles by introducing sequences encoding the ZF1 tag into endogenous loci. Finally, we use ZF1 tagging to establish the site of cdc-42 gene function during a cell invasion event. ZF1 tagging provides a powerful new tool for the analysis of dynamic developmental events.

Invadopodia and basement membrane invasion in vivo.

Over 20 years ago, protrusive, F-actin-based membrane structures, termed invadopodia, were identified in highly metastatic cancer cell lines. Invadopodia penetrate artificial or explanted extracellular matrices in 2D culture conditions and have been hypothesized to facilitate the migration of cancer cells through basement membrane, a thin, dense, barrier-like matrix surrounding most tissues. Despite intensive study, the identification of invadopodia in vivo has remained elusive and until now their possible roles during invasion or even existence have remained unclear. Studies in remarkably different cellular contexts-mouse tumor models, zebrafish intestinal epithelia, and C. elegans organogenesis-have recently identified invadopodia structures associated with basement membrane invasion. These studies are providing the first in vivo insight into the regulation, function, and role of these fascinating subcellular devices with critical importance to both development and human disease.

Traversing the basement membrane in vivo: a diversity of strategies.

The basement membrane is a dense, highly cross-linked, sheet-like extracellular matrix that underlies all epithelia and endothelia in multicellular animals. During development, leukocyte trafficking, and metastatic disease, cells cross the basement membrane to disperse and enter new tissues. Based largely on in vitro studies, cells have been thought to use proteases to dissolve and traverse this formidable obstacle. Surprisingly, recent in vivo studies have uncovered a remarkably diverse range of cellular- and tissue-level strategies beyond proteolysis that cells use to navigate through the basement membrane. These fascinating and unexpected mechanisms have increased our understanding of how cells cross this matrix barrier in physiological and disease settings.