Perspectives of data science in preclinical safety assessment.

The data landscape in preclinical safety assessment is fundamentally changing because of not only emerging new data types, such as human systems biology, or real-world data (RWD) from clinical trials, but also technological advancements in data-processing software and analytical tools based on deep learning approaches. The recent developments of data science are illustrated with use cases for the three factors: predictive safety (new in silico tools), insight generation (new data for outstanding questions); and reverse translation (extrapolating from clinical experience to resolve preclinical questions). Further advances in this field can be expected if companies focus on overcoming identified challenges related to a lack of platforms and data silos and assuring appropriate training of data scientists within the preclinical safety teams.

Entrectinib, a TRK/ROS1 inhibitor with anti-CNS tumor activity: differentiation from other inhibitors in its class due to weak interaction with P-glycoprotein.

Background: Studies evaluating the CNS penetration of a novel tyrosine kinase inhibitor, entrectinib, proved challenging, particularly due to discrepancies across earlier experiments regarding P-glycoprotein (P-gp) interaction and brain distribution. To address this question, we used a novel "apical efflux ratio" (AP-ER) model to assess P-gp interaction with entrectinib, crizotinib, and larotrectinib, and compared their brain-penetration properties.

Methods: AP-ER was designed to calculate P-gp interaction with the 3 drugs in vitro using P-gp-overexpressing cells.

Subcutaneous Site-of-Absorption Study with the Monoclonal Antibody Tocilizumab in Minipigs: Administration Behind Ear Translates Best to Humans.

Minipigs have been proposed as animal model to study the subcutaneous (SC) absorption of monoclonal antibodies (mAb), because they are more translatable to humans than other species. However, the minipig SC tissue structure differs markedly depending on its location. This study explored different SC administration sites for mAb SC administration, to explore which site translates best to humans.

Preliminary pharmacokinetics of tramadol hydrochloride after administration via different routes in male and female B6 mice.

Objective: 1) To determine the pharmacokinetics of tramadol hydrochloride and its active metabolite, O-desmethyltramadol (M1), after administration through different routes in female and male C57Bl/6 mice; 2) to evaluate the stability of tramadol solutions; and 3) to identify a suitable dose regimen for prospective clinical analgesia in B6 mice.

Study Design: Prospective, randomized, blinded, parallel design.

Animals: A total of 18 male and 18 female C57Bl/6 mice (20-30 g).

Extensive metabolism and route-dependent pharmacokinetics of bisphenol A (BPA) in neonatal mice following oral or subcutaneous administration.

Orally administered bisphenol A (BPA) undergoes efficient first-pass metabolism to produce the inactive conjugates BPA-glucuronide (BPA-G) and BPA-sulfate (BPA-S). This study was conducted to evaluate the pharmacokinetics of BPA, BPA-G and BPA-S in neonatal mice following the administration of a single oral or subcutaneous (SC) dose. This study consisted of 3 phases: (1) mass-balance phase in which effective dose delivery procedures for oral or SC administration of (3)H-BPA to postnatal day three (PND3) mice were developed; (2) pharmacokinetic phase during which systemic exposure to total (3)H-BPA-derived radioactivity in female PND3 mice was established; and (3) metabolite profiling phase in which 50 female PND3 pups received either a single oral or SC dose of (3)H-BPA.

Lack of neuropathological changes in rats administered tedizolid phosphate for nine months.

Tedizolid, a novel oxazolidinone antibacterial, was administered to Long Evans rats by oral gavage once daily for up to 9 months at doses near the maximum tolerated dose (MTD) to evaluate for potential neurotoxicity. Mean plasma exposures of tedizolid at the low-, medium-, and high-dose levels (7.5, 15, and 30 mg/kg of body weight/day for males; 2.

LTBP2 and CYP1B1 mutations and associated ocular phenotypes in the Roma/Gypsy founder population.

Primary congenital glaucoma (PCG) is a genetically heterogeneous autosomal recessive disorder, which is an important cause of blindness in childhood. The first known gene, CYP1B1, accounts for a variable proportion of cases in most populations. A second gene, LTBP2, was recently reported in association with a syndrome, in which glaucoma is secondary to lens dislocation.

PON1 and oxidative stress in human sepsis and an animal model of sepsis.

Sepsis is the leading cause of death in critically ill patients. The pathophysiological mechanisms implicated in the development of sepsis and organ failure are complex and involve activation of systemic inflammatory response and coagulation together with endothelial dysfunction. Oxidative stress is a major promoter and mediator of the systemic inflammatory response.

Lactonases with organophosphatase activity: structural and evolutionary perspectives.

Serum paraoxonase (PON1) is well recognized for its ability to hydrolyze arylesters, toxic oxon metabolites of organophosphate insecticides and nerve agents. PON1 is a member of gene family including also PON2 and PON3; however, the later two enzymes have very limited arylesterase and practically no organophosphatase activity. We have established that all three PONs are lactonases/lactonyzing enzymes with overlapping, but also distinct substrate specificity.

Dominant role of paraoxonases in inactivation of the Pseudomonas aeruginosa quorum-sensing signal N-(3-oxododecanoyl)-L-homoserine lactone.

The pathogenic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a key component of P. aeruginosa's quorum-sensing system and regulates the expression of many virulence factors.

Development of an immunoblot assay with infrared fluorescence to quantify paraoxonase 1 in serum and plasma.

Paraoxonase 1 (PON1) requires calcium for activity and is inactivated in the presence of EDTA. Because of this, studies to date have used serum or heparinized plasma for both activity and mass assays of PON1. Whole serum and EDTA plasma were analyzed by SDS-electrophoresis and Western blot using anti-PON1 monoclonal antibody 4C10.

Estrogen esters as substrates for human paraoxonases.

Mammalian paraoxonases (PONs 1, 2 and 3) are a highly conserved family of esterases, with uncertain physiological functions and natural substrates. Here we characterize the ability of purified recombinant human PONs to hydrolyze estrogen esters, a class of compounds previously not known to be PON substrates. PONs hydrolyzed estrogen mono- and diesters at position 3 of the steroid A-ring.

Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities.

The paraoxonase (PON) gene family in humans has three members, PON1, PON2, and PON3. Their physiological role(s) and natural substrates are uncertain. We developed a baculovirus-mediated expression system, suitable for all three human PONs, and optimized procedures for their purification.

Paraoxonase-1 does not reduce or modify oxidation of phospholipids by peroxynitrite.

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated esterase/lactonase implicated to play a role in protection against atherosclerosis. However, the exact mechanism(s) and substrates for PON1 are still uncertain. In this article, we review some of the evidence for PON1's antioxidant activity, as well as our efforts to identify the actual substrates and products for this activity.

Purified human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH.

Purified serum paraoxonase (PON1) had been shown to attenuate the oxidation of LDL in vitro. We critically reevaluated the antioxidant properties of serum PON1 in the in vitro assays initiated with copper or the free radical generator 2,2'-azobis-2-amidinopropane hydrochloride (AAPH). The antioxidant activity of different purified PON1 preparations did not correlate with their arylesterase (AE), lactonase, or phospholipase A2 activities or with the amounts of detergent or protein.

The role of hsp90 in heme-dependent activation of apo-neuronal nitric-oxide synthase.

Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the ubiquitous protein chaperone hsp90. We have shown previously that nNOS expressed in Sf9 cells where endogenous heme levels are low is activated from the apo- to the holo-enzyme by addition of exogenous heme to the culture medium, and this activation is inhibited by radicicol, a specific inhibitor of hsp90 (Billecke, S. S.

Separation and quantitative recovery of mouse serum arylesterase and carboxylesterase activity.

Paraoxonase-1 (PON1) is known to be associated with high density lipoproteins. We optimized buffer conditions to obtain quantitative recovery of PON1 (arylesterase) activity and analyzed the distribution of PON1 in mice using a combination of size-exclusion chromatography and ultracentrifugation. Size-exclusion chromatography of mouse serum separated the esterase activity into two peaks, one overlapping the high density lipoproteins and a second peak of lower molecular weight, consistent with serum carboxylesterase, which accounted for approximately 20% of the total esterase activity of normal mouse serum.

Lactonase and lactonizing activities of human serum paraoxonase (PON1) and rabbit serum PON3.

Human paraoxonase (PON1) was previously shown to hydrolyze over 30 different lactones (cyclic esters). In the present study purified human PON1 was found to catalyze the reverse reaction (lactonization) of a broad range of hydroxy acids. Hydroxy acid lactonization or lactone hydrolysis is catalyzed until equilibrium between the open and closed forms is reached.

Mouse macrophage paraoxonase 2 activity is increased whereas cellular paraoxonase 3 activity is decreased under oxidative stress.

Objective: To determine whether paraoxonases (PONs) are expressed in macrophages and to analyze the oxidative stress effect on their expression and activities.

Methods And Results: We demonstrated the presence (mRNA, protein, activity) of PON2 and PON3 but not PON1 in murine macrophages, whereas in human macrophages, only PON2 was expressed. Under oxidative stress as present in mouse peritoneal macrophages (MPMs) from apoE-deficient (E0) mice as well as in C57BL6 mice, MPMs that were incubated with buthionine sulfoximine, with angiotensin II, with 7-ketocholesterol, or with oxidized phosphatidylcholine, PON2 mRNA levels and lactonase activity toward dihydrocoumarin significantly increased (by 50% to 130%).

Paraoxonase-1 reduces monocyte chemotaxis and adhesion to endothelial cells due to oxidation of palmitoyl, linoleoyl glycerophosphorylcholine.

Objective: High-density lipoprotein (HDL) is postulated to protect against the development of atherosclerosis, in part, by inhibiting the oxidation of low density lipoprotein (LDL) in the sub-endothelial space and thus inhibiting activation of the endothelium. The HDL-associated enzyme, paraoxonase-1, is proposed to be a major protective factor. However, HDL is also prone to oxidation when exposed to peroxynitrite and may therefore, once oxidized, have properties similar to oxidized LDL.

Multiple substrates for paraoxonase-1 during oxidation of phosphatidylcholine by peroxynitrite.

Paraoxonase (PON-1) is a high-density lipoprotein (HDL)-bound enzyme with activity toward multiple substrates. It hydrolyzes organic phosphate and aromatic carboxylic acid esters. It also inhibits accumulation of oxidized phospholipids in plasma lipoproteins by a mechanism yet to be determined.