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BackgroundStudies 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.MethodsAP-ER was designed to calculate P-gp interaction with the 3 drugs in vitro using P-gp–overexpressing cells. Brain penetration was studied in rat plasma, brain, and cerebrospinal fluid (CSF) samples after intravenous drug infusion. Unbound brain concentrations were estimated through kinetic lipid membrane binding assays and ex vivo experiments, while the antitumor activity of entrectinib was evaluated in a clinically relevant setting using an intracranial tumor mouse model.ResultsEntrectinib showed lower AP-ER (1.1–1.15) than crizotinib and larotrectinib (≥2.8). Despite not reaching steady-state brain exposures in rats after 6 hours, entrectinib presented a more favorable CSF-to-unbound concentration in plasma (CSF/Cu,p) ratio (>0.2) than crizotinib and larotrectinib at steady state (both: CSF/Cu,p ~0.03). In vivo experiments validated the AP-ER approach. Entrectinib treatment resulted in strong tumor inhibition and full survival benefit in the intracranial tumor model at clinically relevant systemic exposures.ConclusionsEntrectinib, unlike crizotinib and larotrectinib, is a weak P-gp substrate that can sustain CNS exposure based on our novel in vitro and in vivo experiments. This is consistent with the observed preclinical and clinical efficacy of entrectinib in neurotrophic tropomyosin receptor kinase (NTRK) and ROS1 fusion-positive CNS tumors and secondary CNS metastases.

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. The study assessed the SC absorption of tocilizumab (Actemra®) following administration at several injection sites: Inguinal area, flank, caudal to the ear, and interscapular area, in comparison with an IV group. After SC administration, tocilizumab absorption was most rapid from the inguinal administration site, and slowest after administration behind the ear, with absorption from the other sites in between. Tocilizumab bioavailability was 98.6, 88.3, 74.1, and 86.3% after administration in inguinal area, flank, behind the ear, and interscapular area, as determined by non-compartmental analysis. Fitting of a single first-order absorption rate constant by compartmental analysis was dissatisfactory. A combined fitting of all data was done assuming two different kinds of SC depots, one undergoing fast absorption, the other undergoing a slower absorption. The split between these absorption depots differed across administration sites, with absorption from "fast depot" in inguinal area > flank > interscapular area > behind the ear. Comparisons with clinical data show that tocilizumab PK after SC administration behind the ear translates best to humans, considering both bioavailability and rate of absorption. Whether this translation from minipigs to humans is prototypic for other mAb remains to be confirmed.

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; 2.5, 5, and 10 mg/kg/day for females) were similar between males and females and were 1.8-, 3.9-, and 8.0-fold greater than exposures in patients at the therapeutic dose (200 mg once daily). Evaluated endpoints included survival, clinical observations, body weight, and food consumption. At 1, 3, 6, and 9 months, ophthalmic examinations, functional observational batteries, and locomotor activity measures were conducted, brain weights/sizes were recorded, and perfusion-fixed tissues were collected from 12 rats/sex/group/time point. A detailed morphological assessment was conducted on brain, eyes, optic nerve/tract, spinal cord, peripheral nerves (includes sciatic, sural, tibial, peroneal, trigeminal), and skeletal muscle. At the end of 9 months, less body weight gain was seen in high-dose males (−6.7%) and females (−5.8%) compared with that seen in controls. There were no tedizolid-related adverse neurobehavioral effects or tedizolid-related histopathologic changes in the central/peripheral nervous systems, including the optic nerve. Results of this study indicate that tedizolid was not neurotoxic when administered long term to pigmented rats at doses near the MTD, which were up to 8-fold higher than the human therapeutic exposure.

Entrectinib is a selective and brain penetrant inhibitor with potency against TRKA, TRKB, TRKC, ROS1 and ALK currently in clinical development for the treatment of tumors harboring NTRK or ROS1 gene fusions. In multiple non-clinical models harboring NTRK or ROS1 gene fusions, entrectinib demonstrates potent tumor growth inhibition leading to tumor regressions. Entrectinib achieves exposure in the brain across multiple species with brain-to-plasma concentration ratios of 0.4 in mice, 0.6-1.5 in rats, and 1.4-2.2 in dogs after multiple oral administration. Previously Cook, et al. demonstrated that entrectinib demonstrates anti-tumor efficacy in a BCAN-NTRK1 glioma model, however only testing higher doses. Here we sought to better understand the effective exposures of entrectinib required for pathway inhibition and anti-tumor efficacy through dose-ranging efficacy, pharmacokinetics (PK) and pharmacodynamics (PD) studies using the TPM3-NTRK1 KM12-Luciferase (KM12-Luc) tumor model inoculated subcutaneously or intracranially. In the subcutaneous setting, entrectinib treatment results in significant dose-dependent inhibition of TRKA pathway signaling for up to 12 hours and anti-tumor activity with doses of 5 mg/kg (dosed orally (PO), every day (QD)) and above resulting in strong pathway suppression and tumor regressions. PK analysis demonstrated that an exposure of AUC24h of ≥20 µM.hr was associated with potent TRKA pathway inhibition and tumor growth inhibition. In the intracranial setting, entrectinib similarly demonstrated a dose-dependent effect on inhibition of tumor bioluminescence and enhanced animal survival. Pathway inhibition was observed at all doses of entrectinib with significant activity observed at doses of 5 mg/kg, PO, QD and above. In contrast to tumors inoculated subcutaneously, entrectinib doses of 15 mg/kg, PO, twice daily (BID) or 30 mg/kg, PO, QD were required to strongly inhibit anti-tumor efficacy with the highest dose of 60 mg/kg, PO, BID achieving full suppression of intracranial tumor growth. These data help to establish a model connecting entrectinib drug exposure in circulation and in the brain to pathway suppression and anti-tumor efficacy in an NTRK gene fusion tumor model, which support clinical use of entrectinib in patients with brain NTRK or ROS1 gene fusion tumors. Citation Format: Cecile C. de la Cruz, Thomas Hunsaker, Faye Vazvaei, Dragomir Draganov, Li Yu, Mark Merchant. Determination of the efficacious Entrectinib exposures required for pathway inhibition and anti-tumor activity in a subcutaneous and intracranialTPM3-NTRK1mutant tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3894.

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). Methods Mice were administered 25 mg kg−1 tramadol as a bolus [intravenously (IV), intraperitoneally (IP), subcutaneously (SQ), orally per gavage (OSgavage)] over 25 hours [orally in drinking water (OSwater) or Syrspend SF (OSSyrsp)]. Venous blood was sampled at six predetermined time points over 4 to 31 hours, depending on administration route, to determine tramadol and M1 plasma concentrations (liquid chromatography and tandem mass spectrometry detection). Pharmacokinetic parameters were described using a noncompartmental model. The stability of tramadol in water (acidified and untreated) and Syrspend SF (0.20 mg mL−1) at ambient conditions for 1 week was evaluated. Results After all administration routes, Cmax was >100 ng mL−1 for tramadol and >40 ng mL−1 for M1 (reported analgesic ranges in man) followed by short half-lives (2–6 hours). The mean tramadol plasma concentration after self-administration remained >100 ng mL−1 throughout consumption time. M1 was found in the OSSyrs group only at 7 hours, whereas it was detectable in OSwater throughout administration. Tramadol had low oral bioavailability (26%). Short-lasting side effects were observed only after IV administration. Water and Syrspend SF solutions were stable for 1 week. Conclusions and clinical relevance 1) At the dose administered, high plasma concentrations of tramadol and M1 were obtained, with half-life depending on the administration route. 2) Plasma levels were stable over self-consumption time. 3) Solutions were stable for 1 week at ambient conditions.

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. We report on the molecular and clinical profile of 34 families diagnosed as PCG, all originating from the Roma/Gypsy founder population. Comprehensive sequencing analysis revealed a level of heterogeneity unusual for this population, with five CYP1B1 and one ancestral LTBP2 mutation accounting for ∼70% of patients (25 out of 37) and the remainder still unexplained. Homozygosity for the founder LTBP2 p.R299X mutation resulted in a more severe clinical phenotype and poorer outcome despite a markedly higher number of surgical interventions. The genetically homogeneous group of p.R299X homozygotes showed variable phenotypes (presumably also underlying pathogenetic mechanisms), wherein PCG proper with primary dysgenesis of the trabecular meshwork, and Marfan syndrome-like zonular disease with ectopia lentis and later onset secondary glaucoma are two extremes. The spectrum manifestations may occur in different combinations and have a different evolution even within the same sibship or a single patient. Preliminary observations on compounds with mutations in both CYP1B1-LTBP2 suggest that the observed combinations are of no clinical significance and digenic inheritance is unlikely. We provide a population genetics perspective to explain the allelic heterogeneity, comparing the history and geographic distribution of the two major founder mutations – p.R299X/LTBP2 and p.E387K/CYP1B1.

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. Serum PON1 has been demonstrated in multiple clinical and animal studies to protect against oxidative stress, but also to undergo inactivation upon that condition. We found decreased plasma PON1 activity in patients with sepsis compared to healthy controls or critically ill patients without sepsis; furthermore, in sepsis patients PON1 activity was lower and remained lower in the course of sepsis in the non-survivors compared to the survivors. Plasma PON1 activity was positively correlated with high-density lipoprotein cholesterol and negatively correlated with markers of lipid peroxidation. In an experimental animal model of sepsis, murine cecal ligation and puncture, the time course of plasma PON1 activity was very similar to that found in sepsis patients. Persistently low PON1 activity in plasma was associated with lethal outcome in human and murine sepsis.