Lmx1b is essential for survival of periocular mesenchymal cells and influences Fgf-mediated retinal patterning in zebrafish.

To gain insight into the mechanisms of Lmx1b function during ocular morphogenesis, we have studied the roles of lmx1b.1 and lmx1b.2 during zebrafish eye development. In situ hybridization and characterization of transgenic lines in which GFP is expressed under lmx1b.1 regulatory sequence show that these genes are expressed in periocular tissues and in a pattern conserved with other vertebrates. Anti-sense morpholinos against lmx1b.1 and lmx1b.2 result in defective migration of periocular mesenchymal cells around the eye and lead to apoptosis of these cells. These defects in the periocular mesenchyme are correlated with a failure in fusion of the choroid fissure or in some instances, more severe ventral optic cup morphogenesis phenotypes. Indeed, by blocking the death of the periocular mesenchyme in Lmx1b morphants, optic vesicle morphogenesis is largely restored. Within the retina of lmx1b morphants, Fgf activity is transiently up-regulated and these morphants show defective naso-temporal patterning. Epistasis experiments indicate that the increase in Fgf activity is partially responsible for the ocular anomalies caused by loss of Lmx1b function. Overall, we propose zebrafish lmx1b.1 and lmx1b.2 promote the survival of periocular mesenchymal cells that influence multiple signaling events required for proper ocular development.

Lack of an association between clinical INSTI-related body weight gain and direct interference with MC4 receptor (MC4R), a key central regulator of body weight.

An increasing prevalence of overweight and obesity in people living with HIV has been associated with initiation of antiretroviral therapy with integrase strand transfer inhibitors (INSTIs). An off-target inhibition of the endogenous ligand binding to the human melanocortin 4 receptor (MC4R) has been suggested as a potential mechanism for clinical body weight gain following initiation of dolutegravir, an INSTI. In this study, we interrogated several INSTIs for their capacity for antagonism or agonism of MC4R in an in vitro cell-based assays including at concentrations far exceeding plasma concentrations reached at the recommended dosages. Our results indicate that while INSTIs do exhibit the capacity to antagonize MC4R, this occurs at concentrations well above predicted clinical exposure and is thus an implausible explanation for INSTI-associated weight gain.

The West coast regional safety pharmacology society meeting update: Filling translational gaps in safety assessment.

The Safety Pharmacology Society (SPS) held a West Coast Regional Meeting in Foster City, CA on November 14, 2018 at the Gilead Sciences Inc. site. The meeting was attended by scientists from the pharmaceutical and biotechnology industry, contract research organizations (CROs) and academia. A variety of scientific topics were presented by speakers, covering a broad variety of topics in the fields of safety risk assessment; from pro-arrhythmia and contractility risk evaluation, to models of heart failure and seizure in-a-dish; and discovery sciences; from stem cells and precision medicine, to models of inherited cardiomyopathy and precision cut tissue slices. The present review summarizes the highlights of the presentations and provides an overview of the high level of innovation currently underlying many frontiers in safety pharmacology.

The L:M cone ratio in males of African descent with normal color vision.

Among Caucasian males with normal color vision, long-wavelength-sensitive (L) cones outnumber middle-wavelength-sensitive (M) cones by nearly three to one, on average, and the L and the M cone opsin genes are arrayed on the X-chromosome with the L opsin gene being closest to an upstream enhancer element termed the locus control region (LCR). Interaction between an opsin gene promoter and the LCR is required to mediate normal opsin gene expression, and the relative proximity of the L opsin gene promoter (4,000 base pairs for L compared to 40,000 base pairs for the M opsin gene promoter) has been proposed to endow the L gene with the advantage in competing for interaction with the LCR, thereby accounting for the nearly 3:1 ratio of L:M cones. This proximal advantage hypothesis predicts that the L:M cone ratio will be similar among populations that share the same X-chromosome opsin gene array organization. Here, we tested this hypothesis by examining a sample of males of African descent and found them to have a significantly different average L:M ratio compared to Caucasian males, even though their X-chromosome opsin gene arrays were indistinguishable from arrays in males of Caucasian descent. How these observations might be reconciled is discussed.

Evaluating the human X-chromosome pigment gene promoter sequences as predictors of L:M cone ratio variation.

Men with normal color vision vary widely in the ratio of long- (L) to middle-wavelength sensitive (M) cones. This variation provides opportunities to test models for the mechanism that produces L versus M cones during development. The L and M photopigment genes lie in a tandem array. Each gene has a promoter, and upstream of each array there is a genetic element, termed the locus control region (LCR), that is required for the expression of both L and M pigment genes. During development, for each cell that has been determined to be an L or M cone, it has been proposed that the LCR acts as a stochastic selector which chooses one gene from the array to be expressed. In this model, the L and M promoters compete for contact with the LCR in each photoreceptor. Theoretically, the promoter that, by chance, is the first to successfully form a stable and permanent complex with the LCR commits the cell to a lifetime of exclusive expression of its associated gene. Under this model, it has been suggested that nucleotide differences in the promoters influence their ability to compete in forming a complex with the LCR. Thus, normal variation in L:M cone ratio is predicted to be associated with nucleotide polymorphisms in the promoters. Here we tested this hypothesis by comparing the L and M promoter sequences for 73 males with normal color vision for whom L:M cone ratio estimates had been obtained previously. The M gene promoter sequences were found to be identical for all 73 males and the L gene promoters were identical for 71 out of the 73 males. Two males had mutations where in each case the L promoter differed by one nucleotide substitution compared to normal. Both of the males with promoter mutations had unusual cone ratios which is consistent with the growing body of evidence indicating that the relative ability of the promoters to form a complex with the LCR is a factor in determining cone ratio. However, the vast majority of cone ratio differences were not associated with any difference in the promoter sequence. To explain the high degree of cone ratio variation among normal males, the mechanism that determines whether a cone is L or M must involve genetic elements that have a high degree of genetic polymorphism in the normal population. The results presented here indicate that there are additional genetic components of the mechanism which remain to be identified and incorporated into the present hypotheses.

LC-MS/MS sensitivity enhancement using 2D-SCX/RPLC and its application in the assessment of pharmacokinetics of clonidine in dried blood spots.

BACKGROUND: Dried blood spot (DBS) technology offers distinctive preclinical and clinical advantages primarily ascribed to microscale sampling (e.g., 40-80 µl per time point), and the nature of solid-state samples in filter papers. Logistic benefits in sample collection, storage and shipping also result. However, the effective DBS samples available for bioanalysis are finite, that is, in the order of approximately 1 µl equivalent of plasma (3-mm punch) from a DBS of approximately 15-20 µl whole blood samples. This represents 20- to 100-times fewer samples for bioanalysis compared with a typical plasma assay. It is critical to increase LC-MS/MS sensitivity to accommodate DBS bioanalysis. RESULTS: We developed a 2D strong cation exchange reversed-phase LC-MS/MS (2D-SCX/RPLC-MS/MS) for online enrichment, separation and detection of basic polar compounds, using clonidine hydrochloride as a model compound. Positively charged clonidine was retained and enriched in the first dimensional SCX column even in large volumes, eluted to a second dimensional RP column with ammonium acetate, de-salted with highly aqueous solvent and separated in an analytical RP column. Injection of 100 µl clonidine extract exhibited essentially the same peak shape as that from 1 µl and the response of clonidine increased quantitatively in the range of 1-100 µl. CONCLUSION: The method was successfully employed to analyze clonidine DBS samples from an in-house toxicology study, where clonidine hydrochloride was administered to cynomolgus monkeys to produce hypotensive effects. Of 55 DBS samples collected post-dose, a total of 52 samples were within the curve range of 0.1-50 ng/ml, where valid clonidine PK profiles were obtained. The PK parameters agreed well with the onset of hemodynamic changes measured with implanted miniature telemetry blood pressure transmitters. In comparison, only 21 samples were within the curve range of 2 to 1000 ng/ml from a HILIC-MS/MS method, which limited useful injection volume to 5 µl.

Sensitivity of two noninvasive blood pressure measurement techniques compared to telemetry in cynomolgus monkeys and beagle dogs.

INTRODUCTION: Animals are commonly used in toxicological research for the evaluation of drug effects on the cardiovascular system. Accurate and reproducible determination of blood pressure (BP) in conscious, manually restrained monkeys and dogs is a challenge with current non-invasive cuff techniques. The High Definition Oscillometry (HDO) technique enables real time measurements with immediate visual feedback via PC screen on data validity. HDO measurements are considerably faster with a duration of approximately 8 to 15s than conventional cuff methods that can take several minutes. METHODS: HDO Memo Diagnostic Model Science and Cardell BP Monitor Model 9401 measurements were compared for accuracy and reliability with simultaneously recorded direct blood pressure data captured via radiotelemetry. Six monkeys and six dogs implanted with DSI PCT telemetry transmitters were used; BP data were collected by all methods under manual constraint and compared. Measurements were performed with HDO and Cardell in the presence of a BP lowering drug (hexamethonium bromide). Systolic, diastolic, mean arterial pressure, and pulse rate were determined before, during and following up to 10mg/kg hexamethonium administration via intravenous slow bolus injection. RESULTS: Drug induced hemodynamic changes could be detected in monkeys and dogs with the HDO method but only in dogs with the Cardell method. Correlation coefficients were generally higher for HDO versus Telemetry than Cardell versus Telemetry comparisons, indicating that this novel, non-invasive technique produces reliable blood pressure data and is able to detect drug-induced hemodynamic changes. DISCUSSION: HDO provides an alternative approach for invasive telemetry surgeries to obtain reliable hemodynamic data in animal models for cardiovascular research when invasive techniques are not warranted.

Evaluation of blood pressure measurement using a miniature blood pressure transmitter with jacketed external telemetry in cynomolgus monkeys.

INTRODUCTION: Current techniques used to accurately determine arterial blood pressure (BP) in conscious, unrestrained monkeys require invasive telemetry. This study evaluated the functionality of an implanted miniature telemetry blood pressure transmitter for the collection of BP measurements in conjunction with electrocardiographic measurements using a jacketed external telemetry (JET) system in conscious, unrestrained cynomolgus monkeys. METHODS: Twenty-four animals were surgically implanted with the transmitter in the right femoral artery. Local tolerability to the implant, signal quality, and variability in hemodynamic values were evaluated. On alternate weeks, animals were given single doses of positive control agents to produce hypotensive (clonidine hydrochloride) or hypertensive (L-NAME) effects. Undisturbed telemetry BP data were continuously collected for at least 24h following dosing and analyzed. RESULTS: While exhibiting remarkably high signal quality ( approximately 95% data points retained over 24h of data collection) and moderate variability across study weeks in baseline pulse height measurements (changes as small as < 0 mmHg), nine of 18 transmitters were nonfunctional by 19 weeks post-surgery, most likely due to migration of the catheter out of the artery. In animals given positive control agents, L-NAME induced a statistically significant increase (> or = + 8 mmHg) and clonidine hydrochloride induced a statistically significant decrease (-11 mmHg) in mean arterial pressures. Histological analysis revealed femoral arterial thickening near the sites of implantation. DISCUSSION: These results demonstrate the ability of the miniature BP transmitter, in conjunction with the JET system, to detect small changes in hemodynamic data continuously collected in conscious unrestrained monkeys. Future optimization of the transmitter includes the addition of a suture rib to the transmitter body and increased catheter size to prevent catheter migration out of the artery, the root cause of failed transmitters. The miniature blood pressure transmitter evaluated provides a minimally invasive technique for continuous collection of hemodynamic data in a toxicology study environment.

Topography of long- and middle-wavelength sensitive cone opsin gene expression in human and Old World monkey retina.

The topographical distributions of the relative ratio of long- (L) and middle- (M) wavelength sensitive cone opsin messenger RNA (mRNA) in human and baboon retinas were mapped using real-time polymerase chain reaction. The L:M mRNA ratio increased in a central-to-peripheral gradient in both species, being quite pronounced for humans.