Daniel K. Ness

Exacerbation of Cerebral Amyloid Angiopathy-Associated Microhemorrhage in Amyloid Precursor Protein Transgenic Mice by Immunotherapy Is Dependent on Antibody Recognition of Deposited Forms of Amyloid β

Passive immunization with an antibody directed against the N terminus of amyloid β (Aβ) has recently been reported to exacerbate cerebral amyloid angiopathy (CAA)-related microhemorrhage in a transgenic animal model. Although the mechanism responsible for the deleterious interaction is unclear, a direct binding event may be required. We characterized the binding properties of several monoclonal anti-Aβ antibodies to deposited Aβ in brain parenchyma and CAA. Biochemical analyses demonstrated that the 3D6 and 10D5, two N-terminally directed antibodies, bound with high affinity to deposited forms of Aβ, whereas 266, a central domain antibody, lacked affinity for deposited Aβ. To determine whether 266 or 3D6 would exacerbate CAA-associated microhemorrhage, we treated aged PDAPP mice with either antibody for 6 weeks. We observed an increase in both the incidence and severity of CAA-associated microhemorrhage when PDAPP transgenic mice were treated with the N-terminally directed 3D6 antibody, whereas mice treated with 266 were unaffected. These results may have important implications for future immune-based therapeutic strategies for Alzheimers disease.

Characterization of fumonisin toxicity in orally and intravenously dosed swine

Fumonisin B1 (FB1), a recently identified mycotoxin produced by Fusarium moniliforme in corn, has been shown to cause death in swine due to pulmonary edema, an apparently species specific effect, and to interfere with sphingolipid metabolism in vitro. Here we characterize the toxicity of fumonisins, using female cross-bred swine weighing 6 to 13 kg, and present a hypothesis regarding the mechanism of fumonisin-induced pulmonary edema in swine. FB1 was given daily intravenously (IV) to pig 1 for 9 days for a total of 72 mg (7.9 mg/kg) and to pig 2 for 4 days for a total of 67 mg (4.6 mg/kg). Pig 3 (control) was given saline IV for 9 days. Corn screenings naturally contaminated with FB1 (166 ppm) and FB2 (48 ppm) were fed to pigs 4, 5, and 6, and ground corn was fed to pigs 7 and 8 (controls). Pigs 4 and 7 were killed on day 5; pig 5 was found dead on day 6; and pigs 6 and 8 were killed on day 15. Pigs 4 and 5 had ingested 187 and 176 mg total fumonisins, respectively, while pig 6 had ingested 645 mg. Feed consumption had decreased in pigs fed corn screenings, with an additional sharp decrease prior to onset of clinical signs. Increases in serum liver enzymes, total bilirubin, and cholesterol were present, but electrocardiograms, heart rate, and body temperature were unaffected. Pigs dosed IV with FB1, developed mild intermittent respiratory abnormalities, while those fed screenings developed respiratory distress within 5 days. Mild interstitial pulmonary edema was observed in pig 1. Severe interstitial pulmonary edema, pleural effusion, and increased lung wet/dry weight ratio were observed in pigs 4 and 5. All pigs given fumonisin (either IV or orally) had hepatic changes characterized by hepatocyte disorganization and necrosis; pancreatic acinar cell degeneration was also observed. Ultrastructural changes in orally dosed swine included loss of sinusoidal hepatocyte microvilli; membranous material in hepatic sinusoids; and multilamellar bodies in hepatocytes, Kupffer cells, pancreatic acinar cells and pulmonary macrophages. Pulmonary intravascular macrophages (PIMs) contained large amounts of membranous material. Thus, the target organs of fumonisin in the pig are the lung, liver, and pancreas. At lower doses, slowly progressive hepatic disease is the most prominent feature, while at higher doses, acute pulmonary edema is superimposed on hepatic injury and may cause death. We hypothesize that altered sphingolipid metabolism causes hepatocellular damage resulting in release of membranous material into the circulation. This material is phagocytosed by the PIMs thus triggering the release of mediators which ultimately results in pulmonary edema.

Temporal and dose-response features in swine fed corn screenings contaminated with fumonisin mycotoxins

Fumonisin B1 (FB1), a mycotoxin produced byFusarium moniliforme andF. proliferatum, induces liver damage and pulmonary edema in swine. We examined the temporal and dose-response features of FB1 toxicosis in male weanling crossbred pigs fed nutritionally balanced diets, containing corn screenings naturally contaminated with fumonisins, for 14 days. Total fumonisins (FB1 and FB2) in diets 1 through 6 were assayed at 175, 101, 39, 23, 5, and <1 ppm (below detectable concentrations), respectively. Clinical signs, serum biochemical alterations, and morphologic changes were evaluated. Pigs were weighed, and bled for hematologic and clinical chemistry evaluation on days 5 and 14. They were euthanized on day 14, or earlier if respiratory distress was observed. Respiratory distress developed in 3/5 pigs fed diet 1 between days 4 and 6 due to severe pulmonary edema and pleural effusion. Histologic evidence of hepatic injury was present in all pigs fed diets 1 and 2, 3/5 on diet 3, and 1/5 on diet 4. Serum bilirubin and cholesterol concentrations, gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and arginase (ARG) activities were elevated in pigs fed diets 1 and 2. Based on liver histopathology, the no observed adverse effect level (NOAEL) for fumonisin toxicity in swine was <23 ppm total fumosins for the 14-day period. Based on regression analyses of the clinical chemistry profiles at 14 days, the NOAEL was <12 ppm, with ALP being the most sensitive parameter. In conclusion, pulmonary edema occurred only at the highest fumonisin concentration (175 ppm), while liver damage occurred at much lower concentrations with a NOAEL of <12 ppm.

Leucine-Rich Repeat Kinase 2 (LRRK2)-Deficient Rats Exhibit Renal Tubule Injury and Perturbations in Metabolic and Immunological Homeostasis

Genetic evidence links mutations in the LRRK2 gene with an increased risk of Parkinson’s disease, for which no neuroprotective or neurorestorative therapies currently exist. While the role of LRRK2 in normal cellular function has yet to be fully described, evidence suggests involvement with immune and kidney functions. A comparative study of LRRK2-deficient and wild type rats investigated the influence that this gene has on the phenotype of these rats. Significant weight gain in the LRRK2 null rats was observed and was accompanied by significant increases in insulin and insulin-like growth factors. Additionally, LRRK2-deficient rats displayed kidney morphological and histopathological alterations in the renal tubule epithelial cells of all animals assessed. These perturbations in renal morphology were accompanied by significant decreases of lipocalin-2, in both the urine and plasma of knockout animals. Significant alterations in the cellular composition of the spleen between LRRK2 knockout and wild type animals were identified by immunophenotyping and were associated with subtle differences in response to dual infection with rat-adapted influenza virus (RAIV) and Streptococcus pneumoniae. Ontological pathway analysis of LRRK2 across metabolic and kidney processes and pathological categories suggested that the thioredoxin network may play a role in perturbing these organ systems. The phenotype of the LRRK2 null rat is suggestive of a complex biology influencing metabolism, immune function and kidney homeostasis. These data need to be extended to better understand the role of the kinase domain or other biological functions of the gene to better inform the development of pharmacological inhibitors.

First‐in‐human assessment of PRX002, an anti–α‐synuclein monoclonal antibody, in healthy volunteers

Background: α‐Synuclein is a major component of pathologic inclusions that characterize Parkinsons disease. PRX002 is an antibody that targets α‐synuclein, and its murine parent antibody 9E4 has been shown in preclinical studies to reduce α‐synuclein pathology and to protect against cognitive and motor deteriorations and progressive neurodegeneration in human α‐synuclein transgenic mice. Methods: This first‐in‐human, randomized, double‐blind, placebo‐controlled, phase 1 study assessed the impact of PRX002 administered to 40 healthy participants in 5 ascending‐dose cohorts (n = 8/cohort) in which participants were randomly assigned to receive a single intravenous infusion of study drug (0.3, 1, 3, 10, or 30 mg/kg; n = 6/cohort) or placebo (n = 2/cohort). Results: PRX002 demonstrated favorable safety, tolerability, and pharmacokinetic profiles at all doses tested, with no immunogenicity. No serious adverse events, discontinuations as a result of adverse events, or dose‐limiting toxicities were reported. Serum PRX002 exposure was dose proportional; the average terminal half‐life across all doses was 18.2 days. A significant dose‐dependent reduction in free serum α‐synuclein (unbound to PRX002) was apparent within 1 hour after PRX002 administration, whereas total α‐synuclein (free plus bound) increased dose‐dependently, presumably because of the expected change in kinetics following antibody binding. Conclusions: This study demonstrates that serum α‐synuclein can be safely modulated in a dose‐dependent manner after single intravenous infusions of an anti–α‐synuclein antibody. These findings support continued development of PRX002, including further characterization of its safety, tolerability, pharmacokinetics, and pharmacodynamic effects in the central nervous system in patients with Parkinsons disease.

Quantitative assessment of cumulative carcinogenic risk for multiple genotoxic impurities in a new drug substance.

In pharmaceutical development, significant effort is made to minimize the carcinogenic potential of new drug substances (NDS). This involves appropriate genotoxicity and carcinogenicity testing of the NDS, and understanding the genotoxic potential of its impurities. Current available guidance recommends the use of the threshold of toxicological concern (TTC) for a single impurity where mutagenicity but no carcinogenicity information exists. Despite best efforts, the presence of more than one genotoxic impurity in an NDS may occur at trace levels. This paper repeats the analysis performed by others for a single genotoxic compound, but also uses statistical simulations to assess the impact on cancer risk for a mixture of genotoxic compounds. In summary, with the addition of multiple impurities all controlled to the TTC, an increase in cancer risk was observed. This increase is relatively small when considering the conservative assumptions of the TTC. If structurally similar compounds had an assumed strong correlation (+/-10-fold from the first randomly selected impurity) in cancer potency, the resulting cancer risk was not negatively impacted. Findings based on probabilistic analysis here can be very useful in making appropriate decisions about risk management of multiple genotoxic impurities measured in the final drug substance.

Pharmacological inhibition of polo like kinase 2 (PLK2) does not cause chromosomal damage or result in the formation of micronuclei.

Polo like kinase 2 (PLK2) phosphorylates α-synuclein and is considered a putative therapeutic target for Parkinsons disease. Several lines of evidence indicate that PLK2 is involved with proper centriole duplication and cell cycle regulation, inhibition of which could impact chromosomal integrity during mitosis. The objectives of the series of experiments presented herein were to assess whether specific inhibition of PLK2 is genotoxic and determine if PLK2 could be considered a tractable pharmacological target for Parkinsons disease. Several selective PLK2 inhibitors, ELN 582175 and ELN 582646, and their inactive enantiomers, ELN 582176 and ELN 582647, did not significantly increase the number of micronuclei in the in vitro micronucleus assay. ELN 582646 was administered to male Sprague Dawley rats in an exploratory 14-day study where flow cytometric analysis of peripheral blood identified a dose-dependent increase in the number of micronucleated reticulocytes. A follow-up investigative study demonstrated that ELN 582646 administered to PLK2 deficient and wildtype mice significantly increased the number of peripheral micronucleated reticulocytes in both genotypes, suggesting that ELN 582646-induced genotoxicity is not through the inhibition of PLK2. Furthermore, significant reduction of retinal phosphorylated α-synuclein levels was observed at three non-genotoxic doses, additional data to suggest that pharmacological inhibition of PLK2 is not the cause of the observed genotoxicity. These data, in aggregate, indicate that PLK2 inhibition is a tractable CNS pharmacological target that does not cause genotoxicity at doses and exposures that engage the target in the sensory retina.

Discovery of a novel [3.2.1] benzo fused bicyclic sulfonamide-pyrazoles as potent, selective and efficacious γ-secretase inhibitors

Structure-activity relationship (SAR) of a novel, potent and metabolically stable series of benzo [3.2.1] bicyclic sulfonamide-pyrazoles as γ-secretase inhibitors are described. Compounds that are efficacious in reducing the cortical Aβx-40 levels in FVB mice via oral dose, as well as those with high selectivity over Notch, are highlighted.

Safety and Tolerability of Multiple Ascending Doses of PRX002/RG7935, an Anti–α-Synuclein Monoclonal Antibody, in Patients With Parkinson Disease: A Randomized Clinical Trial

Importance Aggregated &agr;-synuclein is believed to be central to the pathogenesis of Parkinson disease (PD). PRX002/RG7935 (PRX002) is a humanized monoclonal antibody designed to target aggregated forms of &agr;-synuclein, thereby inhibiting neuron-to-neuron transfer of presumed pathogenic forms of &agr;-synuclein, potentially resulting in neuronal protection and slowing disease progression. Objective To evaluate the safety and tolerability of multiple intravenous infusions of PRX002 in patients with idiopathic PD. Design, Setting, and Participants Multicenter, randomized, double-blind, placebo-controlled, multiple ascending-dose trial at 8 US study centers from July 2014 to September 2016. Eligible participants were aged 40 to 80 years with mild to moderate idiopathic PD (Hoehn and Yahr stages 1-3). Interventions Participants were enrolled into 6 ascending-dose cohorts and randomly assigned to receive PRX002 (0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg, or 60 mg/kg) or placebo. Participants received 3 intravenous infusions every 4 weeks of PRX002 or placebo and were monitored during a 24-week observational period. Main Outcomes and Measures Safety and tolerability assessments included physical and neurological examinations, laboratory tests, vital signs, and adverse events. Pharmacokinetic parameters included maximum PRX002 concentration, area under the curve, and half-life. Results Of the 80 participants, most were white (97.5%; n = 78) and male (80%; n = 64); median (SD) age was 58 (8.4) years. PRX002 was generally safe and well tolerated; no serious or severe PRX002-related treatment-emergent adverse events (TEAEs) were reported. The TEAEs experienced by at least 5% of patients receiving PRX002, irrespective of relatedness to study drug, were constipation (9.1%; n = 5), infusion reaction (7.3%; n = 4), diarrhea (5.5%; n = 3), headache (5.5%; n = 3), peripheral edema (5.5%; n = 3), post–lumbar puncture syndrome (5.5%; n = 3), and upper respiratory tract infection (5.5%; n = 3). No antidrug antibodies were detected. Serum PRX002 levels increased in an approximately dose-proportional manner; mean terminal elimination half-life was similar across all doses (10.2 days). Rapid dose- and time-dependent mean reductions from baseline vs placebo in free serum &agr;-synuclein levels of up to 97% were seen after a single infusion at the highest dose (F78,284 = 1.66; P = .002), with similar reductions after 2 additional infusions. Mean cerebrospinal fluid PRX002 concentration increased with PRX002 dose and was approximately 0.3% relative to serum across all dose cohorts. Conclusions and Relevance Single and multiple doses of PRX002 were generally safe and well tolerated and resulted in robust binding of peripheral &agr;-synuclein and dose-dependent increases of PRX002 in cerebrospinal fluid, reaching cerebrospinal fluid concentrations that may be expected to engage extracellular aggregated &agr;-synuclein in the brain. Findings support the design of an ongoing phase 2 clinical study (NCT03100149). Trial Registration ClinicalTrials.gov Identifier: NCT02157714

Pharmacokinetic and pharmacodynamic analysis of a novel gamma secretase inhibitor in a non-transgenic A-beta mouse efficacy model

feed male mice for 3 months either with a [DHAþ] (0.1% in the chow) or [DHA-] diet, prior Ab injection. Cognitive, histological and biochemical studies were performed to evaluate the outcomes of such a supplementation. Results: Dietary DHA was shown to lead to significantly increased levels in the hippocampus and frontal cortex. This DHA-enrichment was associated with the protection of synaptic proteins. Both parameters were strongly correlated with the learning and memory capacities and were implicated in the prevention of Ab oligomer-induced impairments. Conclusions: Altogether, our data demonstrate the interest of our non-transgenic mouse model for the evaluation of AD-preventing approaches and strongly imply DHA as a pivotal ingredient in the view of designing well-ageing diets.