Selective Cannabinoid 2 Receptor Agonists as Potential Therapeutic Drugs for the Treatment of Endotoxin-Induced Uveitis.

(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists, RO6871304, and RO6871085, as well as a CB2R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB2R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R > CB1R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC50s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB2R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases.

NF-κB activation in astrocytes drives a stage-specific beneficial neuroimmunological response in ALS.

Astrocytes are involved in non-cell-autonomous pathogenic cascades in amyotrophic lateral sclerosis (ALS); however, their role is still debated. We show that astrocytic NF-κB activation drives microglial proliferation and leukocyte infiltration in the SOD1 (G93A) ALS model. This response prolongs the presymptomatic phase, delaying muscle denervation and decreasing disease burden, but turns detrimental in the symptomatic phase, accelerating disease progression. The transition corresponds to a shift in the microglial phenotype showing two effects that can be dissociated by temporally controlling NF-κB activation. While NF-κB activation in astrocytes induced a Wnt-dependent microglial proliferation in the presymptomatic phase with neuroprotective effects on motoneurons, in later stage, astrocyte NF-κB-dependent microglial activation caused an accelerated disease progression. Notably, suppression of the early microglial response by CB2R agonists had acute detrimental effects. These data identify astrocytes as important regulators of microglia expansion and immune response. Therefore, stage-dependent microglia modulation may be an effective therapeutic strategy in ALS.

Process and scaling parameters for wet media milling in early phase drug development: A knowledge based approach.

Wet media milling is a well-established unit operation for the manufacturing of suspension formulations during early phase pharmaceutical development. However, knowledge about the kinetics of particle breakage is limited, although the impact of hydro-mechanical process parameters on the mean particle size of finished suspensions has been thoroughly investigated. We performed in this work milling trials with two different compounds on two milling devices with different mechanical design and volume scale. We analyzed our data in terms of a kinetic milling model where we included the milling speed as an explicit process parameter in addition to the process duration. We show, that the milling kinetics can be fairly well predicted for a wide range of these operating parameters. The proposed milling model may therefore be useful for rational process planning and scale-up considerations in the industrial setting.

Time domain NMR as a new process monitoring method for characterization of pharmaceutical hydrates.

Hydrates are of great pharmaceutical relevance and even though they have been characterized thoroughly by various analytical techniques, there is barely literature available on molecular mobility of the hydrate water studied by NMR relaxation in the time domain. The aim of this work was to examine the possibility of differentiating hydration states of drugs by 1H time domain NMR (TD-NMR) regarding spin-spin and spin-lattice relaxation times (T2 and T1) using benchtop equipment. Caffeine and theophylline were selected as model compounds and binary mixtures of hydrate to anhydrate were analyzed for each drug using a spin echo and inversion recovery pulse sequence. It was possible to extract a signal that was specific for the water in the hydrates so that differentiation from anhydrous solid forms was enabled. Excellent calibrations were obtained for quantitative analysis of hydrate/anhydrate mixtures and predicted water contents were in good agreement with water amounts determined in desiccator sorption experiments. TD-NMR was therefore found to be a suitable new technique to characterize pharmaceutical hydrates in a non-invasive and hence sample-sparing manner. Quantification of the hydrate content in pharmaceutical mixtures appears highly attractive for product development and process monitoring. TD-NMR provides here a valuable and complementary technique to established process analytics, such as for example Raman spectroscopy.

Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity.

The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

Novel Triazolopyrimidine-Derived Cannabinoid Receptor 2 Agonists as Potential Treatment for Inflammatory Kidney Diseases.

The cannabinoid receptor 2 (CB2) system is described to modulate various pathological conditions, including inflammation and fibrosis. A series of new heterocyclic small-molecule CB2 receptor agonists were identified from a high-throughput screen. Lead optimization gave access to novel, highly potent, and selective (over CB1) triazolopyrimidine derivatives. A preliminary structure-activity relationship was established, and physicochemical properties in this compound class were significantly improved toward better solubility, lipophilicity, and microsomal stability. An optimized triazolopyrimidine derivative, (3S)-1-[5-tert-butyl-3-[(1-cyclopropyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol (39), was tested in a kidney ischemia-reperfusion model, in which it showed efficacy at a dose of 10 mg kg(-1) (p.o.). A significant depletion of the three measured kidney markers indicated a protective role of CB2 receptor activation toward inflammatory kidney damage. Compound 39 was also protective in a model of renal fibrosis. Oral treatment with 39 at 3 mg kg(-1) per day significantly decreased the amount of fibrosis by ∼ 40% which was induced by unilateral ureter obstruction.

Time domain 1H NMR as a new method to monitor softening of gelatin and HPMC capsule shells.

Defined mechanical properties are an essential requirement for any pharmaceutical dosage form and this is particularly important in the case of liquid-filled capsules. Changes in the mechanical properties may be induced by exposure of the capsules to humidity or by a shift of the water equilibrium that typically occurs when hydrophilic or amphiphilic fill masses are used, for example, in self-emulsifying drug delivery systems. This study aims to characterize the softening of empty hard gelatin and hydroxypropyl methylcellulose (HPMC) capsules by means of mechanical tests, a Bareiss hardness test, and a stiffness test using a texture analysis method. A benchtop time domain NMR method is applied in addition to characterize the physico-chemical state of water in the capsule shells and to correlate this with the results of the mechanical tests. Hardness and stiffness measurements resulted in corresponding values, showing a softening for both capsule materials in a humid environment, which was most pronounced beyond 60% relative humidity. The capsules made of gelatin exhibited in general higher stiffness and hardness values compared to the HPMC capsules. The physico-chemical state of water in the capsule shells, as probed by a time domain NMR method, was interpreted in terms of a population balance model. Three different water populations were identified that differ in their molecular mobility, as indicated by their characteristic spin-lattice relaxation times, T1. The most loosely bound water fraction dominated in the capsule shells in the range beyond 60% relative humidity. Numerical correlation of the data led to a heuristic equation between the NMR-derived fraction of loosely bound water in the capsule shells and their mechanical stiffness and hardness. Adequate models were obtained for both capsule types, gelatin, and HPMC. Mechanical measurements of pharmaceutical capsules are generally destructive and time consuming. Testing is usually performed in an analytical laboratory, off-line from the manufacturing process, and involves only a small number of samples. Based on the here presented correlation between mechanical stiffness measurements and benchtop time domain NMR data, the latter method may be used as a nondestructive alternative for mechanical testing. This study also opens the possibility to investigate liquid-filled capsules and to establish a process analytical technology (PAT) during manufacturing.