Improved automated spot counting and modeling with bias correction.

A complete workflow was presented for estimating the concentration of microorganisms in biological samples by automatically counting spots that represent viral plaque forming units (PFU) bacterial colony forming units (CFU), or spot forming units (SFU) in images, and modeling the counts. The workflow was designed for processing images from dilution series but can also be applied to stand-alone images. The accuracy of the methods was greatly improved by adding a newly developed bias correction method. When the spots in images are densely populated, the probability of spot overlapping increases, leading to systematic undercounting. In this paper, this undercount issue was addressed in an empirical way. The proposed empirical bias correction method utilized synthetic images with known spot sizes and counts as a training set, enabling the development of an effective bias correction function using a thin-plate spline model. Its application focused on the bias correction for the automated spot counting algorithm LoST proposed by Lin et al. Simulation results demonstrated that the empirical bias correction significantly improved spot counts, reducing bias for both fixed and random spot sizes and counts.

Niraparib Shows Superior Tissue Distribution and Efficacy in a Prostate Cancer Bone Metastasis Model Compared with Other PARP Inhibitors.

Patients with prostate cancer whose tumors bear deleterious mutations in DNA-repair pathways often respond to PARP inhibitors. Studies were conducted to compare the activity of several PARP inhibitors in vitro and their tissue exposure and in vivo efficacy in mice bearing PC-3M-luc-C6 prostate tumors grown subcutaneously or in bone. Niraparib, olaparib, rucaparib, and talazoparib were compared in proliferation assays, using several prostate tumor cell lines and in a cell-free PARP-trapping assay. PC-3M-luc-C6 cells were approximately 12- to 20-fold more sensitive to PARP inhibition than other prostate tumor lines, suggesting that these cells bear a DNA damage repair defect. The tissue exposure and efficacy of these PARP inhibitors were evaluated in vivo in PC-3M-luc-C6 subcutaneous and bone metastasis tumor models. A steady-state pharmacokinetic study in PC-3M-luc-C6 tumor-bearing mice showed that all of the PARP inhibitors had favorable subcutaneous tumor exposure, but niraparib was differentiated by superior bone marrow exposure compared with the other drugs. In a PC-3M-luc-C6 subcutaneous tumor efficacy study, niraparib, olaparib, and talazoparib inhibited tumor growth and increased survival to a similar degree. In contrast, in the PC-3M-luc-C6 bone metastasis model, niraparib showed the most potent inhibition of bone tumor growth compared with the other therapies (67% vs. 40%-45% on day 17), and the best survival improvement over vehicle control [hazard ratio (HR), 0.28 vs. HR, 0.46-0.59] and over other therapies (HR, 1.68-2.16). These results show that niraparib has superior bone marrow exposure and greater inhibition of tumor growth in bone, compared with olaparib, rucaparib, and talazoparib.

The Impact of Experimental and Calculated Error on the Performance of Affinity Predictions.

The accurate prediction of binding affinity between protein and small molecules with free energy methods, particularly the difference in binding affinities via relative binding free energy calculations, has undergone a dramatic increase in use and impact over recent years. The improvements in methodology, hardware, and implementation can deliver results with less than 1 kcal/mol mean unsigned error between calculation and experiment. This is a remarkable achievement and beckons some reflection on the significance of calculation approaching the accuracy of experiment. In this article, we describe a statistical analysis of the implications of variance (standard deviation) of both experimental and calculated binding affinities with respect to the unknown true binding affinity. We reveal that plausible ratios of standard deviation in experiment and calculation can lead to unexpected outcomes for assessing the performance of predictions. The work extends beyond the case of binding free energies to other affinity or property prediction methods.

Motor cortical excitability and paired-associative stimulation-induced plasticity in amnestic mild cognitive impairment and Alzheimer's disease.

OBJECTIVE: Synaptopathy including alterations of synaptic plasticity (long-term potentiation, LTP) may precede neurodegeneration in Alzheimer's disease (AD). We studied LTP-like corticospinal plasticity induced by paired-associative stimulation (PASLTP) in AD and its prodromal stage, amnestic mild cognitive impairment (aMCI). METHODS: 15 AD and 15 aMCI patients, and 23 demographically matched healthy controls (HC) were included. Resting motor threshold (RMT) and stimulus intensity needed to evoke motor evoked potentials (MEP) of 1 mV (SI1mV) were obtained as single-pulse transcranial magnetic stimulation (TMS) measures of corticospinal excitability in a hand muscle at baseline, followed by PASLTP using standard methodology. MEP amplitude change after PASLTP normalized to baseline was defined as plasticity effect. All measures were repeated in two visits for examining test-retest reliability. RESULTS: SI1mV were lower in aMCI compared to HC, while there was no difference between AD and HC. RMT and SI1mV showed excellent test-retest reliability in all groups. PASLTP indiscriminately did not induce LTP-like plasticity in any of the groups, and expressed poor test-retest reliability. CONCLUSIONS: aMCI shows corticospinal hyperexcitability, consistent with glutamatergic excitotoxicity in early-stage AD. Possible abnormalities of LTP-like plasticity could not be reliably tested with the standard PASLTP protocol due to massive inter-subject variability even in HC, and poor test-retest reliability. SIGNIFICANCE: Findings indicate corticospinal hyperexcitability in prodromal AD, and reliability of single-pulse TMS measures for identifying such abnormality. In contrast, the standard PASLTP protocol may not be suitable for assessing LTP-like motor cortical plasticity, given its overall nil effect and poor test-retest reliability.

Translational evaluation of novel selective orexin-1 receptor antagonist JNJ-61393215 in an experimental model for panic in rodents and humans.

Orexin neurons originating in the perifornical and lateral hypothalamic area project to anxiety- and panic-associated neural circuitry, and are highly reactive to anxiogenic stimuli. Preclinical evidence suggests that the orexin system, and particularly the orexin-1 receptor (OX1R), may be involved in the pathophysiology of panic and anxiety. Selective OX1R antagonists thus may constitute a potential new treatment strategy for panic- and anxiety-related disorders. Here, we characterized a novel selective OX1R antagonist, JNJ-61393215, and determined its affinity and potency for human and rat OX1R in vitro. We also evaluated the safety, pharmacokinetic, and pharmacodynamic properties of JNJ-61393215 in first-in-human single- and multiple-ascending dose studies conducted. Finally, the potential anxiolytic effects of JNJ-61393215 were evaluated both in rats and in healthy men using 35% CO2 inhalation challenge to induce panic symptoms. In the rat CO2 model of panic anxiety, JNJ-61393215 demonstrated dose-dependent attenuation of CO2-induced panic-like behavior without altering baseline locomotor or autonomic activity, and had minimal effect on spontaneous sleep. In phase-1 human studies, JNJ-61393215 at 90 mg demonstrated significant reduction (P < 0.02) in CO2-induced fear and anxiety symptoms that were comparable to those obtained using alprazolam. The most frequently reported adverse events were somnolence and headache, and all events were mild in severity. These results support the safety, tolerability, and anxiolytic effects of JNJ-61393215, and validate CO2 exposure as a translational cross-species experimental model to evaluate the therapeutic potential of novel anxiolytic drugs.

Pharmacodynamics of atabecestat (JNJ-54861911), an oral BACE1 inhibitor in patients with early Alzheimer's disease: randomized, double-blind, placebo-controlled study.

BACKGROUND: ß-Secretase enzyme (BACE) inhibition has been proposed as a priority treatment mechanism for Alzheimer's disease (AD), but treatment initiation may need to be very early. We present proof of mechanism of atabecestat (also known as JNJ-54861911), an oral BACE inhibitor for the treatment of AD, in Caucasian and Japanese populations with early AD who do not show signs of dementia. METHODS: In two similarly designed phase I studies, a sample of amyloid-positive elderly patients comprising 45 Caucasian patients with early AD diagnosed as preclinical AD (n = 15, Clinical Dementia Rating [CDR] = 0) or with mild cognitive impairment due to AD (n = 30, CDR = 0.5) and 18 Japanese patients diagnosed as preclinical AD (CDR-J = 0) were randomized 1:1:1 to atabecestat 10 or 50 mg or placebo (n = 6-8/treatment) daily for 4 weeks. Safety, pharmacokinetics (PK), and pharmacodynamics (PD) (i.e., reduction of cerebrospinal fluid [CSF] amyloid beta 1-40 [Aß1-40] levels [primary endpoint] and effect on other AD biomarkers) of atabecestat were evaluated. RESULTS: In both populations, atabecestat was well tolerated and characterized by linear PK and high central nervous system penetrance of unbound drug. Atabecestat significantly reduced CSF Aß1-40 levels from baseline at day 28 in both the 10-mg (67-68%) and 50-mg (87-90%) dose groups compared with placebo. For Caucasians with early AD, the least squares mean differences (95% CI) were - 69.37 (- 72.25; - 61.50) and - 92.74 (- 100.08; - 85.39), and for Japanese with preclinical AD, they were - 62.48 (- 78.32; - 46.64) and - 80.81 (- 96.13; - 65.49), respectively. PK/PD model simulations confirmed that once-daily 10 mg and 50 mg atabecestat can attain 60-70% and 90% Aß1-40 reductions, respectively. The trend of the reduction was similar across the Aß1-37, Aß1-38, and Aß1-42 fragments in both atabecestat dose groups, consistent with Aß1-40. CSF amyloid precursor protein fragment (sAPPß) levels declined from baseline, regardless of patient population, whereas CSF sAPPα levels increased compared with placebo. There were no relevant changes in either CSF total tau or phosphorylated tau 181P over a 4-week treatment period. CONCLUSIONS: JNJ-54861911 at 10 and 50 mg daily doses after 4 weeks resulted in mean CSF Aß1-40 reductions of 67% and up to 90% in both Caucasian and Japanese patients with early stage AD, confirming results in healthy elderly adults. TRIAL REGISTRATION: ALZ1005: ClinicalTrials.gov, NCT01978548. Registered on 7 November 2013. ALZ1008: ClinicalTrials.gov, NCT02360657. Registered on 10 February 2015.

A multiple-dose double-blind randomized study to evaluate the safety, pharmacokinetics, pharmacodynamics and analgesic efficacy of the TRPV1 antagonist JNJ-39439335 (mavatrep).

BACKGROUND AND AIMS: This double-blind (DB), randomized, placebo-controlled, sequential-group, multiple-ascending dose, phase 1 study evaluated safety, pharmacokinetics and pharmacodynamics of JNJ-39439335 in healthy men (part 1), and in participants with knee osteoarthritis (part 2). METHODS: Both parts 1 and 2 consisted of screening (upto 21 days), 21-day DB treatment phase [eight participants/group: JNJ-39439335 (part 1: 2-50 mg; part 2: 10-50 mg): n=6; placebo: n=2] and follow-up (total study duration ~10 weeks). RESULTS: Plasma concentrations and systemic exposure of JNJ-39439335 increased in slightly higher than dose-proportional fashion (steady-state reached by day 14). Renal excretion of JNJ-39439335 was negligible. Marked dose-related increases in pharmacodynamic heat pain assessments were observed in JNJ-39439335-treated participants, which persisted throughout the treatment with no signs of tolerance with repeated dosing. No effect on pharmacodynamic cold pain or mechanical pain assessments were seen. Effects on pharmacodynamic capsaicin-induced flare assessments in JNJ-39439335-treated participants versus placebo were consistent with effects observed with single-dose, and did not demonstrate tolerance with multiple dosing. In participants with knee osteoarthritis, significant improvements versus placebo were observed in a stair-climbing-induced pain model. All JNJ-39439335-treated participants reported ≥1 treatment-emergent adverse events (TEAE); most common (≥50% incidence) TEAEs in part 1 were feeling hot (79%), thermohypoesthesia (71%), paresthesia (58%) and feeling cold (50%), and in part 2, were minor thermal burns (50%). CONCLUSIONS: JNJ-39439335 (doses 2-50 mg) was well-tolerated, and associated with acceptable multiple-dose pharmacokinetic profile. JNJ-39439335 demonstrated sustained pharmacodynamic effects (heat pain perception, heat pain latency, capsaicin-induced flare), and an efficacy signal in participants with osteoarthritis pain. IMPLICATIONS: Given the efficacy signal observed and the unique safety profile, larger phase 2 studies are needed to better understand the potential of JNJ-39439335 in the treatment of chronic pain. Analgesic efficacy of lower doses administered over a longer period of time and improved patient counseling techniques to reduce the minor thermal burns can be explored to minimize the adverse events.