GraphCpG: imputation of single-cell methylomes based on locus-aware neighboring subgraphs.

MOTIVATION: Single-cell DNA methylation sequencing can assay DNA methylation at single-cell resolution. However, incomplete coverage compromises related downstream analyses, outlining the importance of imputation techniques. With a rising number of cell samples in recent large datasets, scalable and efficient imputation models are critical to addressing the sparsity for genome-wide analyses. RESULTS: We proposed a novel graph-based deep learning approach to impute methylation matrices based on locus-aware neighboring subgraphs with locus-aware encoding orienting on one cell type. Merely using the CpGs methylation matrix, the obtained GraphCpG outperforms previous methods on datasets containing more than hundreds of cells and achieves competitive performance on smaller datasets, with subgraphs of predicted sites visualized by retrievable bipartite graphs. Besides better imputation performance with increasing cell number, it significantly reduces computation time and demonstrates improvement in downstream analysis. AVAILABILITY AND IMPLEMENTATION: The source code is freely available at https://github.com/yuzhong-deng/graphcpg.git.

The Absolute Bioavailability and Absorption, Metabolism, and Excretion of Ipatasertib, a Potent and Highly Selective Protein Kinase B (Akt) Inhibitor.

Ipatasertib (GDC-0068) is a potent, highly selective, small-molecule inhibitor of protein kinase B (Akt) being developed by Genentech/Roche as a single agent and in combination with other therapies for the treatment of cancers. To fully understand the absorption, metabolism, and excretion of ipatasertib in humans, an open-label study using 14C-radiolabeled ipatasertib was completed to characterize the absolute bioavailability (period 1) and mass balance and metabolite profiling (period 2). In period 1, subjects were administered a 200 mg oral dose of ipatasertib followed by an 80 µg (800 nCi) intravenous dose of [14C]-ipatasertib. In period 2, subjects received a single oral dose containing approximately 200 mg (100 µCi) [14C]-ipatasertib. In an integrated analytical strategy, accelerator mass spectrometry was applied to measure the 14C microtracer intravenous pharmacokinetics in period 1 and fully profile plasma radioactivity in period 2. The systemic plasma clearance and steady-state volume of distribution were 98.8 L/h and 2530 L, respectively. The terminal half-lives after oral and intravenous administrations were similar (26.7 and 27.4 hours, respectively) and absolute bioavailability of ipatasertib was 34.0%. After a single oral dose of [14C]-ipatasertib, 88.3% of the administered radioactivity was recovered with approximately 69.0% and 19.3% in feces and urine, respectively. Radioactivity in feces and urine was predominantly metabolites with 24.4% and 8.26% of dose as unchanged parent, respectively; indicating that ipatasertib had been extensively absorbed and hepatic metabolism was the major route of clearance. The major metabolic pathway was N-dealkylation mediated by CYP3A, and minor pathways were oxidative by cytochromes P450 and aldehyde oxidase. SIGNIFICANCE STATEMENT: The study provided definitive information regarding the absolute bioavailability and the absorption, metabolism, and excretion pathways of ipatasertib, a potent, novel, and highly selective small-molecule inhibitor of protein kinase B (Akt). An ultrasensitive radioactive counting method, accelerator mass spectrometry was successfully applied for 14C-microtracer absolute bioavailability determination and plasma metabolite profiling.

Exposure-Effect Relationships in Established Rat Adjuvant-Induced and Collagen-Induced Arthritis: A Translational Pharmacokinetic-Pharmacodynamic Analysis.

The ability of rodent immune-mediated arthritis models to quantitatively predict therapeutic activity of antiarthritis agents is poorly understood. Two commonly used preclinical models of arthritis are adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) in rats. The objective of the current study is to investigate the relationship between efficacy in AIA and CIA in rats, and clinical efficacy in rheumatoid arthritis patients using translational pharmacokinetic-pharmacodynamic (PK-PD) analysis. A range of doses of indomethacin (a nonsteroidal anti-inflammatory drug), and three disease-modifying antirheumatic drugs (DMARDs), methotrexate, etanercept, and tofacitinib, were evaluated in AIA and CIA rats. Dexamethasone was included in this study as a positive control. The area under the ankle diameter-time profile (AUCankle) and ankle histopathology summed scores (AHSS) were used as efficacy endpoints for activity against disease symptoms (joint inflammation) and disease progression (joint damage), respectively. Translational PK-PD analysis was performed to rank order preclinical efficacy endpoints at clinically relevant concentrations. For each drug tested, inhibition of AUCankle and AHSS scores was generally comparable in both magnitude and rank order. Overall, based on both AUCankle and the AHSS inhibition, the rank ordering of preclinical activity for the DMARDs evaluated was tofacitinib > etanercept ≥ methotrexate. This ranking of preclinical efficacy was consistent with reported clinical efficacy. Of interest, indomethacin showed equal or often better efficacy than the three DMARDs evaluated on inhibiting AHSS despite having limited ability to prevent joint damage clinically in patients. The translational value of performing PK-PD analysis of arthritis models in rats is discussed.

Catalytic Cleavage of Disulfide Bonds in Small Molecules and Linkers of Antibody-Drug Conjugates.

In cells, catalytic disulfide cleavage is an essential mechanism in protein folding and synthesis. However, detailed enzymatic catalytic mechanism relating cleavage of disulfide bonds in xenobiotics is not well understood. This study reports an enzymatic mechanism of cleavage of disulfide bonds in xenobiotic small molecules and antibody conjugate (ADC) linkers. The chemically stable disulfide bonds in substituted disulfide-containing pyrrolobenzodiazepine (PBD, pyrrolo[2,1-c][1,4]benzodiazepine) monomer prodrugs in presence of glutathione or cysteine were found to be unstable in incubations in whole blood of humans and rats. It was shown the enzymes involved were thioredoxin (TRX) and glutaredoxin (GRX). For a diverse set of drug-linker conjugates, we determined that TRX in the presence of TRX-reductase and NADPH generated the cleaved products that are consistent with catalytic disulfide cleavage and linker immolation. GRX was less rigorously studied; in the set of compounds studied, its role in the catalytic cleavage was also confirmed. Collectively, these in vitro experiments demonstrate that TRX as well as GRX can catalyze the cleavage of disulfide bonds in both small molecules and linkers of ADCs.

Exploration of Pyrrolobenzodiazepine (PBD)-Dimers Containing Disulfide-Based Prodrugs as Payloads for Antibody-Drug Conjugates.

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.

Absorption, metabolism and excretion of cobimetinib, an oral MEK inhibitor, in rats and dogs.

1. The absorption, metabolism and excretion of cobimetinib, an allosteric inhibitor of MEK1/2, was characterized in mass balance studies following single oral administration of radiolabeled (14C) cobimetinib to Sprague-Dawley rats (30 mg/kg) and Beagle dogs (5 mg/kg). 2. The oral dose of cobimetinib was well absorbed (81% and 71% in rats and dogs, respectively). The maximal plasma concentrations for cobimetinib and total radioactivity were reached at 2-3 h post-dose. Drug-derived radioactivity was fully recovered (∼90% of the administered dose) with the majority eliminated in feces via biliary excretion (78% of the dose for rats and 65% for dogs). The recoveries were nearly complete after the first 48 h following dosing. 3. The metabolic profiles indicated extensive metabolism of cobimetinib prior to its elimination. For rats, the predominant metabolic pathway was hydroxylation at the aromatic core. Lower exposures for cobimetinib and total radioactivity were observed in male rats compared with female rats, which was consistent to in vitro higher clearance of cobimetinib for male rats. For dogs, sequential oxidative reactions occurred at the aliphatic portion of the molecule. Though rat metabolism was well-predicted in vitro with liver microsomes, dog metabolism was not. 4. Rats and dogs were exposed to the two major human circulating Phase II metabolites, which provided relevant metabolite safety assessment. In general, the extensive sequential oxidative metabolism in dogs, and not the aromatic hydroxylation in rats, was more indicative of the metabolism of cobimetinib in humans.

Hominoid-specific enzyme GLUD2 promotes growth of IDH1R132H glioma.

Somatic mutation of isocitrate dehydrogenase 1 (IDH1) is now recognized as the most common initiating event for secondary glioblastoma, a brain tumor type arising with high frequency in the frontal lobe. A puzzling feature of IDH1 mutation is the selective manifestation of glioma as the only neoplasm frequently associated with early postzygotic occurrence of this genomic alteration. We report here that IDH1(R132H) exhibits a growth-inhibitory effect that is abrogated in the presence of glutamate dehydrogenase 2 (GLUD2), a hominoid-specific enzyme purportedly optimized to facilitate glutamate turnover in human forebrain. Using murine glioma progenitor cells, we demonstrate that IDH1(R132H) exerts a growth-inhibitory effect that is paralleled by deficiency in metabolic flux from glucose and glutamine to lipids. Examining human gliomas, we find that glutamate dehydrogenase 1 (GLUD1) and GLUD2 are overexpressed in IDH1-mutant tumors and that orthotopic growth of an IDH1-mutant glioma line is inhibited by knockdown of GLUD1/2. Strikingly, introduction of GLUD2 into murine glioma progenitor cells reverses deleterious effects of IDH1 mutation on metabolic flux and tumor growth. Further, we report that glutamate, a substrate of GLUD2 and a neurotransmitter abundant in mammalian neocortex, can support growth of glioma progenitor cells irrespective of IDH1 mutation status. These findings suggest that specialization of human neocortex for high glutamate neurotransmitter flux creates a metabolic niche conducive to growth of IDH1 mutant tumors.

Absorption, Metabolism, Excretion, and the Contribution of Intestinal Metabolism to the Oral Disposition of [14C]Cobimetinib, a MEK Inhibitor, in Humans.

The pharmacokinetics, metabolism, and excretion of cobimetinib, a MEK inhibitor, were characterized in healthy male subjects (n = 6) following a single 20 mg (200 µCi) oral dose. Unchanged cobimetinib and M16 (glycine conjugate of hydrolyzed cobimetinib) were the major circulating species, accounting for 20.5% and 18.3% of the drug-related material in plasma up to 48 hours postdose, respectively. Other circulating metabolites were minor, accounting for less than 10% of drug-related material in plasma. The total recovery of the administered radioactivity was 94.3% (±1.6%, S.D.) with 76.5% (±2.3%) in feces and 17.8% (±2.5%) in urine. Metabolite profiling indicated that cobimetinib had been extensively metabolized with only 1.6% and 6.6% of the dose remaining as unchanged drug in urine and feces, respectively. In vitro phenotyping experiments indicated that CYP3A4 was predominantly responsible for metabolizing cobimetinib. From this study, we concluded that cobimetinib had been well absorbed (fraction absorbed, Fa = 0.88). Given this good absorption and the previously determined low hepatic clearance, the systemic exposures were lower than expected (bioavailability, F = 0.28). We hypothesized that intestinal metabolism had strongly attenuated the oral bioavailability of cobimetinib. Supporting this hypothesis, the fraction escaping gut wall elimination (Fg) was estimated to be 0.37 based on F and Fa from this study and the fraction escaping hepatic elimination (Fh) from the absolute bioavailability study (F = Fa × Fh × Fg). Physiologically based pharmacokinetics modeling also showed that intestinal clearance had to be included to adequately describe the oral profile. These collective data suggested that cobimetinib was well absorbed following oral administration and extensively metabolized with intestinal first-pass metabolism contributing to its disposition.

Discovery of oxa-sultams as RORc inverse agonists showing reduced lipophilicity, improved selectivity and favorable ADME properties.

Modification of the δ-sultam ring of RORc inverse agonist 2 led to the discovery of more polar oxa-sultam 65. The less lipophilic inverse agonist (65) displayed high potency in a biochemical assay, which translated into inhibition of IL-17 production in human peripheral blood mononuclear cells. The successful reduction of lipophilicity of this new analog gave rise to additional improvements in ROR selectivity and aqueous kinetic solubility, as well as reduction in plasma protein binding, while maintaining high cellular permeability.

Identification of N-sulfonyl-tetrahydroquinolines as RORc inverse agonists.

A high-throughput screen of the Genentech/Roche compound collection using a retinoic acid receptor-related orphan receptor C (RORc, RORγ, or NR1F3) biochemical assay revealed a N-sulfonyl-tetrahydroquinoline hit. Herein, we describe the hit-to-lead optimization and structure-activity relationships of these tetrahydroquinoline RORc inverse agonists. Through iterative synthesis and analog design, we identified compounds with improved biochemical RORc inverse agonist activity and RORc cellular potencies. These improved N-sulfonyl-tetrahydroquinoline compounds also exhibited selectivity for RORc over other nuclear receptors.

Discovery of imidazo[1,5-a]pyridines and -pyrimidines as potent and selective RORc inverse agonists.

The nuclear receptor (NR) retinoic acid receptor-related orphan receptor gamma (RORγ, RORc, or NR1F3) is a promising target for the treatment of autoimmune diseases. RORc is a critical regulator in the production of the pro-inflammatory cytokine interleukin-17. We discovered a series of potent and selective imidazo[1,5-a]pyridine and -pyrimidine RORc inverse agonists. The most potent compounds displayed >300-fold selectivity for RORc over the other ROR family members, PPARγ, and NRs in our cellular selectivity panel. The favorable potency, selectivity, and physiochemical properties of GNE-0946 (9) and GNE-6468 (28), in addition to their potent suppression of IL-17 production in human primary cells, support their use as chemical biology tools to further explore the role of RORc in human biology.

Assessment of the hepatic CYP reductase null mouse model and its potential application in drug discovery.

CYP Oxidoreductase (Por) is the essential electron donor for all CYP enzymes and is responsible for the activation of CYP. The Taconic Hepatic CYP Reductase Null (HRN) mouse model possesses a targeted mutation that results in liver-specific deletion of the Por gene thereby resulting in a disruption of CYP metabolism in the liver. The objectives of these studies were to further characterize the HRN mouse using probe drugs metabolized by CYP. In addition, tumor exposure in xenograft tumor bearing HRN immune-compromised (nude) mice was also determined. In HRN mice following intravenous (iv) administration of midazolam, clearance (CL) was reduced by ∼ 80% compared to wild-type mice (WT). After oral administration, the AUC of midazolam was increased by ∼ 20-fold in HRN mice compared to WT mice; this greater effect suggests that hepatic first pass plays a role in the oral CL of midazolam. A 50% and an 80% decrease in CL were also observed in HRN mice following iv administration of docetaxel and theophylline, respectively, compared to WT mice. In addition, a 2- to 3-fold increase in tumor concentrations of G4222, a tool compound, were observed in tumor bearing HRN nude mice compared to tumor bearing nude WT mice. The observations from these experiments demonstrate that, for compounds that are extensively metabolized by hepatic CYP, the HRN mouse model could potentially be valuable for evaluating in vivo efficacy of tool compounds in drug discovery where high hepatic CL and low exposure may prevent in vivo evaluation of a new chemical entity.

Reduction in lipophilicity improved the solubility, plasma-protein binding, and permeability of tertiary sulfonamide RORc inverse agonists.

Using structure-based drug design principles, we identified opportunities to reduce the lipophilicity of our tertiary sulfonamide RORc inverse agonists. The new analogs possessed improved RORc cellular potencies with >77-fold selectivity for RORc over other nuclear receptors in our cell assay suite. The reduction in lipophilicity also led to an increased plasma-protein unbound fraction and improvements in cellular permeability and aqueous solubility.

A reversed sulfonamide series of selective RORc inverse agonists.

The identification of a new series of RORc inverse agonists is described. Comprehensive structure-activity relationship studies of this reversed sulfonamide series identified potent RORc inverse agonists in biochemical and cellular assays which were also selective against a panel of nuclear receptors. Our work has contributed a compound that may serve as a useful in vitro tool to delineate the complex biological pathways involved in signalling through RORc. An X-ray co-crystal structure of an analogue with RORc has also provided useful insights into the binding interactions of the new series.

Absolute bioavailability and effect of formulation change, food, or elevated pH with rabeprazole on cobimetinib absorption in healthy subjects.

Cobimetinib is a potent and highly selective inhibitor of MEK1/2. Since cobimetinib exhibited absorption variability in cancer patients, a series of single-dose studies in healthy subjects were conducted to determine absolute bioavailability and elucidate potential effects of formulation, food, and elevated gastric pH on cobimetinib bioavailability. Three crossover trials were performed with a 20 mg cobimetinib oral dose: absolute bioavailability using a 2 mg intravenous infusion (n = 13), relative bioavailability of tablets versus capsules and food effect (n = 20), and drug interaction with a proton pump inhibitor (20 mg of rabeprazole daily for 5 days prior to cobimetinib administration; n = 20). Absolute bioavailability of cobimetinib was 46.2% (24.2, CV %), likely due to metabolism rather than incomplete absorption. The mean systemic clearance of cobimetinib was low (11.7 L/h [28.2, CV %]). Administration of cobimetinib tablets with a high-fat meal delayed drug absorption (prolonged tmax) but had no statistically significant effect on cobimetinib exposure (Cmax and AUC0-∞). Tablet and capsule formulations of cobimetinib showed comparable exposures. Cobimetinib exhibited delayed absorption (tmax) in the presence of rabeprazole, with no statistically significant effects on drug exposure (Cmax and AUC0-∞) in the fasted state. In conclusion, cobimetinib oral absorption was not affected by change in formulation, food, or elevated gastric pH.

Preclinical disposition of GDC-0973 and prospective and retrospective analysis of human dose and efficacy predictions.

[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone (GDC-0973) is a potent and highly selective inhibitor of mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase (ERK) 1/2 (MEK1/2), a MAPK kinase that activates ERK1/2. The objectives of these studies were to characterize the disposition of GDC-0973 in preclinical species and to determine the relationship of GDC-0973 plasma concentrations to efficacy in Colo205 mouse xenograft models. The clearance (CL) of GDC-0973 was moderate in mouse (33.5 ml · min(-1) · kg(-1)), rat (37.9 ± 7.2 ml · min(-1) · kg(-1)), and monkey (29.6 ± 8.5 ml · min(-1) · kg(-1)). CL in dog was low (5.5 ± 0.3 ml · min(-1) · kg(-1)). The volume of distribution across species was large, 6-fold to 15-fold body water; half-lives ranged from 4 to 13 h. Protein binding in mouse, rat, dog, monkey, and human was high, with percentage unbound, 1 to 6%. GDC-0973-related radioactivity was rapidly and extensively distributed to tissues; however, low concentrations were observed in the brain. In rats and dogs, [(14)C]GDC-0973 was well absorbed (fraction absorbed, 70-80%). The majority of [(14)C]GDC-0973-related radioactivity was recovered in the bile of rat (74-81%) and dog (65%). The CL and volume of distribution of GDC-0973 in human, predicted by allometry, was 2.9 ml · min(-1) · kg(-1) and 9.9 l/kg, respectively. The predicted half-life was 39 h. To characterize the relationship between plasma concentration of GDC-0973 and tumor growth inhibition, pharmacokinetic-pharmacodynamic modeling was applied using an indirect response model. The KC(50) value for tumor growth inhibition in Colo205 xenografts was estimated to be 0.389 µM, and the predicted clinical efficacious dose was ∼10 mg. Taken together, these data are useful in assessing the disposition of GDC-0973, and where available, comparisons with human data were made.

Novel nanoparticles formulation for cassette dosing via intravenous injection in rats for high throughput pharmacokinetic screening and potential applications.

In recent years, one of the biggest challenges for pharmaceutical industry is to increase the speed of finding new medicines while at the same time controlling the ever rising cost of drug discovery and development. In order to increase the speed at which drug candidates are identified, high throughput assays (HTS) have been developed and have been widely implemented in the pharmaceutical industry. Cassette (or N-in-1) dosing for pharmacokinetic (PK) evaluation is the process of generating in vivo PK data in a higher throughput manner by dosing multiple compounds to individual animals. However, due to generally poor solubility of compounds being tested, high percentages of organic solvents are often used in the formulation vehicle in order to solubilize compounds for cassette studies. Utilization of high organic content in formulation vehicles can result in unwanted side effects and animal tolerability issues. The current study evaluates the suitability of using nanoparticles in an aqueous suspension for cassette IV dosing. Nanoparticles of 10 poorly soluble marketed drugs covering a wide range of clearances were prepared using an electrospray device and evaluated. PXRD, TGA and particle size data were obtained in order to ensure the quality for in vivo evaluation. Phosphate buffered saline (PBS) was used as the vehicle in IV cassette study using nanoparticles and pharmacokinetic estimates from this study were comparable to those from a traditional high organic formulation approach. The use of nanoparticles in an aqueous suspension formulation was demonstrated to be appropriate for cassette dosing.

Single- and multiple-dose pharmacokinetics, potential for CYP3A inhibition, and food effect in patients with cancer and healthy subjects receiving ipatasertib.

PURPOSE: To examine the single- and multiple-dose pharmacokinetics (PK), CYP3A inhibition potential of ipatasertib, and effect of food on PK of ipatasertib in patients with refractory solid tumors and a dedicated food effect assessment in healthy subjects. METHODS: The Phase I dose-escalation study enrolled patients with solid tumors in a standard 3 + 3 design with a 1 week washout after the first dose, followed by once-daily dosing on a 3-week-on/1-week-off schedule. In the expansion cohort, the effect of ipatasertib on CYP3A substrate (midazolam) was assessed by examining the change in midazolam exposure when dosed in the absence and presence of steady-state ipatasertib at 600 mg. The effect of food on ipatasertib PK was studied with ipatasertib administered in fed or fasted state (6 patients from Phase I patient study and 18 healthy subjects from the dedicated food effect study). RESULTS: Ipatasertib was generally well tolerated at doses up to 600 mg given daily for 21 days. Ipatasertib showed rapid absorption (tmax, 0.5-3 h), was dose-proportional over a range of 200-800 mg, had a median half-life (range) of 45.0 h (27.8-66.9 h), and had approximately two-fold accumulation following once-daily dosing. Midazolam exposure (AUC0-∞) increased by 2.2-fold in the presence of ipatasertib. PK was comparable in subjects administered ipatasertib in a fed or fasted state. CONCLUSION: Ipatasertib exhibited rapid absorption and was dose-proportional over a broad dose range. Ipatasertib appeared to be a moderate CYP3A inhibitor when administered at 600 mg and could be administered with or without food in clinical studies. TRAIL REGISTRATION: NCT01090960 (registered March 23, 2010); NCT02536391 (registered August 31, 2015).

Effect of Hepatic Impairment on Cobimetinib Pharmacokinetics: The Complex Interplay Between Physiological Changes and Drug Characteristics.

Cobimetinib is a kinase inhibitor indicated for use in combination with vemurafenib for treatment of unresectable/metastatic melanoma with specific BRAF mutations. Cobimetinib is extensively metabolized in liver; thus, patients with hepatic impairment (HI) might have increased cobimetinib exposure. In this study, we investigated the impact of HI on the pharmacokinetics (PK) and safety of cobimetinib. Subjects with normal hepatic function and mild to severe HI were enrolled. All subjects received a single oral dose of 10 mg cobimetinib, and serial blood samples were collected at specified times. Cobimetinib PK in subjects with mild and moderate HI was similar to that in those with normal liver function. However, subjects with severe HI, on average, showed ∼30% lower total AUC0-∞ and ∼2-fold higher unbound AUC0-∞ compared with those with normal hepatic function. These exposure differences can be explained by lower albumin levels observed in subjects with severe HI, the strong correlation between albumin level and the unbound fraction and the general PK variability of cobimetinib. In addition, previous studies with cobimetinib showed a lack of an exposure-response relationship for efficacy and safety. Therefore, collectively, our results suggest that the starting dose for patients with hepatic impairment can be the same as that for those with normal hepatic function.

Ocular phenotypes in a mouse model of impaired glucocerebrosidase activity.

Mutations in the GBA1 gene encoding glucocerebrosidase (GCase) are linked to Gaucher (GD) and Parkinson's Disease (PD). Since some GD and PD patients develop ocular phenotypes, we determined whether ocular phenotypes might result from impaired GCase activity and the corresponding accumulation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph) in the Gba1D409V/D409V knock-in (Gba KI/KI; "KI") mouse. Gba KI mice developed age-dependent pupil dilation deficits to an anti-muscarinic agent; histologically, the iris covered the anterior part of the lens with adhesions between the iris and the anterior surface of the lens (posterior synechia). This may prevent pupil dilation in general, beyond an un-responsiveness of the iris to anti-muscarinics. Gba KI mice displayed atrophy and pigment dispersion of the iris, and occlusion of the iridocorneal angle by pigment-laden cells, reminiscent of secondary open angle glaucoma. Gba KI mice showed progressive thinning of the retina consistent with retinal degeneration. GluSph levels were increased in the anterior and posterior segments of the eye, suggesting that accumulation of lipids in the eye may contribute to degeneration in this compartment. We conclude that the Gba KI model provides robust and reproducible eye phenotypes which may be used to test for efficacy and establish biomarkers for GBA1-related therapies.