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The blood–brain barrier (BBB) is often a limiting factor for getting drugs in the brain. Bypassing the BBB by intranasal (IN), or also called nose to brain (NTB), route is an interesting and frequently investigated concept for brain drug delivery. However, despite the body of evidence for IN drug delivery in literature over the last decades, reproducibility and interpretation of animal data remain challenging. The objective of this project was to assess the feasibility and value of a standardized IN screening model in rats for the evaluation of direct brain delivery. A chemically diverse set of commercial and internal small molecules were tested in the in vivo model with different doses and/or formulations. Data were analyzed using different ways of ratio calculations: blood concentration at time of sacrifice, total exposure in blood (area under the curve, AUC) and the brain or olfactory bulb concentrations. The IN route was compared to another parenteral route to decide if there is potential direct brain transport. The results show that blood and tissue concentrations and ratios are highly variable and not always reproducible. Potential direct brain delivery was concluded for some compounds, however, sometimes depending on the analysis: using blood levels at sacrifice or AUC could lead to different conclusions. We conclude that a screening model for the evaluation of direct brain transport of small molecules is very difficult to achieve and a conclusion based on a limited number of animals with this variability is questionable.

The β-site amyloid precursor protein cleaving enzyme 1 (BACE1, also known as β-secretase) is a promising target for the treatment of Alzheimer's disease. A pKa lowering approach over the initial leads was adopted to mitigate hERG inhibition and P-gp efflux, leading to the design of 6-CF3 dihydrothiazine 8 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-cyanopicolinamide). Optimization of 8 led to the discovery of 15 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-(fluoromethoxy)pyrazine-2-carboxamide) with an excellent balance of potency, hERG inhibition, P-gp efflux, and metabolic stability. Oral administration of 8 elicited robust Aβ reduction in dog even at 0.16 mg/kg. Reflecting the reduced hERG inhibitory activity, no QTc prolongation was observed at high doses. The potential for reactive metabolite formation of 15 was realized in a nucleophile trapping assay using [14 C]-KCN in human liver microsomes. Utilizing covalent binding (CVB) in human hepatocytes and the maximum projected human dosage, the daily CVB burden of 15 was calculated to be at an acceptable value of below 1 mg/day. However, hepatotoxicity was observed when 15 was subjected to a two-week tolerance study in dog, which prevented further evaluation of this compound.

In previous studies, the introduction of electron withdrawing groups to 1,4-oxazine BACE1 inhibitors reduced the pKa of the amidine group, resulting in compound 2 that showed excellent in vivo efficacy, lowering Aβ levels in brain and CSF. However, a suboptimal cardiovascular safety margin, based on QTc prolongation, prevented further progression. Further optimization resulted in the replacement of the 2-fluoro substituent by a CF3-group, which reduced hERG inhibition. This has led to compound 3, with an improved cardiovascular safety margin and sufficiently safe in GLP toxicity studies to progress into clinical trials.

Introduction Clinical observations are routinely used to address drug-induced behavioural changes in dogs. To deal with the limitations of this methodology, we evaluated Actiwatch-Mini® as a tool to monitor continuously locomotor activity in dogs and to provide objective parameters that could be linked to behavioural changes after compound administration. Methods Eight Beagles were equipped with Actiwatch-Mini®. As a reference drug, a PDE2-inhibitor was administered daily for six days at doses known to cause minor or severe behavioural changes. Results While traditional observation showed no behavioural changes - except sedation - dogs receiving 0.5 mg/kg/day, showed significant increases in % immobile time (+15.8%) and mean length of immobile phases (+1.2 min) but no change in number of immobile phases (+2.2). At 1 mg/kg/day, light to severe changes in behaviour were present. Actiwatch-Mini® recorded an increase in mean length of immobile phases (+1.9 min) and a decrease in mean number of immobile phases (−7.4) in the first four hours after each dosing while total % immobile time was not significantly increased (+4.9%). Discussion Actiwatch-Mini® was able to detect changes in immobility parameters in dogs dosed with a PDE2-inhibitor while no or only minor behavioural changes were observed. The system can be used for continuously monitoring the activity of dogs, to provide objective scores for evaluation of activity. It provides an inexpensive and low labour-intensive method for detecting changes in activity and possible early indications of drug-induced behavioural changes.

Genetic evidence points to deposition of amyloid-β (Aβ) as a causal factor for Alzheimer's disease. Aβ generation is initiated when β-secretase (BACE1) cleaves the amyloid precursor protein. Starting with an oxazine lead 1, we describe the discovery of a thiazine-based BACE1 inhibitor 5 with robust Aβ reduction in vivo at low concentrations, leading to a low projected human dose of 14 mg/day where 5 achieved sustained Aβ reduction of 80% at trough level.

Beta-secretase is the first cleavage enzyme of amyloid-β protein precursor (AβPP) in the amyloidogenic pathway, leading to the formation of the plaque forming Amyloid-β (Aβ)1-42 peptide. BACE (beta-site AβPP cleaving enzyme) 1 inhibition is therefore considered to be a promising disease modifying therapy for Alzheimer's disease. An early assessment of the in vivo activity of BACE inhibitors was done in dogs since AβPP processing is the same as in humans and this species easily enables longitudinal cerebrospinal fluid (CSF) sampling. Aβ changes in CSF compared to baseline are used to evaluate target engagement of the compounds. Levels of Aβ1-37, Aβ1-38, Aβ1-40, and Aβ1-42 in CSF are measured with immunoassay (Mesoscale electrochemiluminescence technology) and with an ultra high-performance liquid chromatography mass spectrometry (UPLC-MS/MS). Two experimental BACE inhibitors were evaluated. With the immunoassay, a dose dependent decrease is observed for all four Aβ peptides. Measurements with the UPLC-MS/MS are in line with the immunoassay for Aβ1-37, Aβ1-38, and Aβ1-40, however, for Aβ1-42, differences are sometimes observed when comparing to changes seen in the other peptides with UPLC-MS/MS and with immunoassay results. Generally lower concentrations are measured with immunoassay. The reason for these differences is still unknown. Aβ1-42 is more prone to form aggregates compared to the other peptides. One hypothesis could be that while the immunoassay only measures free Aβ, bound and aggregated Aβ peptides are at least partially dissolved with the UPLC-MS/MS method, since acetonitrile is added to the CSF samples. This increases variability in the concentration of Aβ peptide measured with UPLC-MS/MS, especially for Aβ1-42, potentially masking the compound effect on Aβ1-42 levels.

Nalmefene is an opioid antagonist which as a once-a-day tablet formulation has recently been approved for reducing ethanol intake in alcoholic subjects. In order to address the compliance issue in this patient population, a number of potential nalmefene prodrugs were synthesized with the aim of providing a formulation that could provide plasma drug concentrations in the region of 0.5–1.0 ng/mL for a one-month period when dosed intramuscular to dogs or minipigs. In an initial series of studies, three different lipophilic nalmefene derivatives were evaluated: the palmitate (C16), the octadecyl glutarate diester (C18-C5) and the decyl carbamate (CB10). They were administered intramuscularly to dogs in a sesame oil solution at a dose of 1 mg-eq. nalmefene/kg. The decyl carbamate was released relatively quickly from the oil depot and its carbamate bond was too stable to be used as a prodrug. The other two derivatives delivered a fairly constant level of 0.2–0.3 ng nalmefene/mL plasma for one month and since there was no significant difference between these two, the less complex palmitate monoester was chosen to demonstrate that dog plasma nalmefene concentrations were dose-dependent at 1, 5 and 20 mg-eq. nalmefene/kg. In a second set of experiments, the effect of the chain length of the fatty acid monoester promoieties was examined. The increasingly lipophilic octanoate (C8), decanoate (C10) and dodecanoate (C12) derivatives were evaluated in dogs and in minipigs, at a dose of 5 mg-eq. nalmefene/kg and plasma nalmefene concentrations were measured over a four-week period. The pharmacokinetic profiles were very similar in both species with Cmax decreasing and Tmax increasing with increasing fatty acid chain length and the target plasma concentrations (0.5–1.0 ng/mL over a month-long period) were achieved with the dodecanoate (C12) prodrug. These data therefore demonstrate that sustained plasma nalmefene concentrations can be achieved in both dog and minipig using nalmefene prodrugs and that the pharmacokinetic profile of nalmefene can be tuned by varying the length of the alkyl group.

The next-generation human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase inhibitor rilpivirine (TMC278) was administered in rats and dogs as single intramuscular (IM) or subcutaneous (SC) injections, formulated as a 200-nm nanosuspension. The plasma pharmacokinetics, injection site concentrations, disposition to lymphoid tissues, and tolerability were evaluated in support of its potential use as a once-monthly antiretroviral agent in humans. Rilpivirine plasma concentration-time profiles showed sustained and dose-proportional release over 2 months in rats and over 6 months in dogs. The absolute bioavailability approached 100%, indicating a complete release from the depot, in spite of rilpivirine concentrations still being high at the injection site(s) 3 months after administration in dogs. For both species, IM administration was associated with higher initial peak plasma concentrations and a more rapid washout than SC administration, which resulted in a stable plasma-concentration profile over at least 6 weeks in dogs. The rilpivirine concentrations in the lymph nodes draining the IM injection site exceeded the plasma concentrations by over 100-fold 1 month after administration, while the concentrations in the lymphoid tissues decreased to 3- to 6-fold the plasma concentrations beyond 3 months. These observations suggest uptake of nanoparticles by macrophages, which generates secondary depots in these lymph nodes. Both SC and IM injections were generally well tolerated and safe, with observations of a transient inflammatory response at the injection site. The findings support clinical investigations of rilpivirine nanosuspension as a long-acting formulation to improve adherence during antiretroviral therapy and for preexposure prophylaxis.

A dog model was developed to test the capacity of boosters for antiretroviral medication. Two dogs were implanted with a modified constant-flow Codman 3000® infusion pump, adapted to release viscous solutions of darunavir (TMC114) at a constant rate of 25 mg/dog/day in the venous blood stream. Booster candidates were given by oral gavage for at least 4 days up to maximum 7 days in cross-over fashion, separated by a wash-out period of minimum 1 week. The booster candidates were tested at doses of 20 and/or 40 mg/kg/day: blood sampling for determination of the boosting effect was performed on the last day of booster administration. The model allowed to (1) compare the boosting ratio of these booster candidates based on the exposure (determination of the area under the curve (AUC) of darunavir in presence versus absence of the booster candidate), (2) detect delay in boosting activity by evaluation of the shift of Cmax of darunavir following booster administration versus the Cmax of the booster candidate) and (3) calculate the intrinsic booster capacity, by correcting for the systemic exposure of booster candidate by normalizing the booster ratio for the booster's AUC. The latter parameter (intrinsic booster capacity) allows to determine the booster's metabolic contribution in inhibiting the metabolism of antiretroviral medication (most likely via inhibition of CYP3A4), minimizing the impact of potential effects of the booster at the level of the gastro-intestinal tract.

1,4-Oxazines are presented, which show good in vitro inhibition in enzymatic and cellular BACE1 assays. We describe lead optimization focused on reducing the amidine pKa while optimizing interactions in the BACE1 active site. Our strategy permitted modulation of properties such as permeation and especially P-glycoprotein efflux. This led to compounds which were orally bioavailable, centrally active, and which demonstrated robust lowering of brain and CSF Aβ levels, respectively, in mouse and dog models. The amyloid lowering potential of these molecules makes them valuable leads in the search for new BACE1 inhibitors for the treatment of Alzheimer’s disease.

The design and the synthesis of several chemical subclasses of imidazole containing γ-secretase modulators (GSMs) is described. Conformational restriction of pyridone 4 into bicyclic pyridone isosteres has led to compounds with high in vitro and in vivo potency. This has resulted in the identification of benzimidazole 44a as a GSM with low nanomolar potency in vitro. In mouse, rat, and dog, this compound displayed the typical γ-secretase modulatory profile by lowering Aβ42 and Aβ40 levels combined with an especially pronounced increase in Aβ38 and Aβ37 levels while leaving the total levels of amyloid peptides unchanged.

BACE1 is a key enzyme for amyloid-β (Aβ) production, and an attractive therapeutic target in Alzheimer's disease (AD). Here we report that BACE1 inhibitors have distinct effects on neuronal Aβ metabolism, inducing a unique pattern of secreted Aβ peptides, analyzed in cell media from amyloid precursor protein (APP) transfected cells and in cerebrospinal fluid (CSF) from dogs by immunoprecipitation-mass spectrometry, using several different BACE1 inhibitors. Besides the expected reductions in Aβ1-40 and Aβ1-42, treatment also changed the relative levels of several other Aβ isoforms. In particular Aβ1-34 decreased, while Aβ5-40 increased, and these changes were more sensitive to BACE1 inhibition than the changes in Aβ1-40 and Aβ1-42. The effects on Aβ5-40 indicate the presence of a BACE1 independent pathway of APP degradation. The described CSF Aβ pattern may be used as a pharmacodynamic fingerprint to detect biochemical effects of BACE1-therapies in clinical trials, which might accelerate development of novel therapies.