Characterization of 2-AG as a potential biomarker

The endocannabinoid (eCB) system is an intricate and well conserved biological system located both the central nervous system (CNS) and peripherally. As a result, it has been implicated in a vast array of physiological processes such as emotion regulation, feeding behavior, pain sensation, reward behaviour, glucose metabolism, memory, and sleep.
The endocannabinoid system is comprised of the G-protein coupled receptors (GPCRs) cannabinoid receptor type 1 (CB1R) and cannabinoid receptor type 2 (CB2R), their endogenous ligands 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA or anandamide), as well as the enzymes responsible for the synthesis and degradation of these molecules. AEA and 2-AG are two endocannabinoids whose role has been extensively investigated in the context of CNS disorder and they are synthesized from membrane-derived lipid precursors coupled to their release, while degradation occurs via fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. CB1R and CB2R are G-protein-coupled receptors and are found in almost every major system throughout the body. Specifically, CB1R is highly expressed in the CNS, particularly in cortex, hippocampus, amygdala, basal ganglia and cerebellum, whereas CB2R is mostly present on immune cells. Also, the eCB system plays critical modulatory role in synaptic plasticity and homeostasis, and has been implicated in learning, memory formation and mood regulation.
In fact, accumulating evidence supports cross-talk between eCB signaling and the synaptic growth factor brain derived neurotrophic factor (BDNF) which is recognized as a key player in brain neurogenesis and synaptic plasticity. Together, several lines of research support targeting the eCB system in the treatment of neuropsychiatric disease in general, and particularly so for affective disorders such as anxiety and depression.
Agonists, antagonists or inverse agonists, that indiscriminately or/and potently activate or inhibit the function of CB1Rs, can interfere with normal endocannabinoid-mediated function in non-target cells, with untoward clinical effects as a result. As opposed to targeting CB1Rs directly, selective inhibition of the degradation enzymes FAAH or MAGL are expected to increase endocannabinoid concentrations, which may enable more subtle modulation of the eCB system. Noteworthy, FAAH and MAGL inhibition are dependent upon 2 different mechanisms. 2-AG, the major substrate for MAGL, is present in significantly greater concentrations (i.e., 2 orders of magnitude higher) in the brain as compared to anandamide; notably, 2-AG is the most abundant endogenous agonist of cannabinoid receptors in the body. JNJ-69095897 is a selective, reversible, centrally active, non-competitive inhibitor of MAGL
currently in preclinical development. Several clinical studies are currently being planned in different human populations.
Pharmacodynamic biomarkers are crucial in first-in-human (FIH) studies since they can guide dose selection and safety decisions based on demonstration of target engagement. However, methodological and technical issues related to the biomarker may unknowingly influence its validity. For example, circadian rhythm or external factors such as sleep, or diet may act as confounders by influencing biomarker concentrations. Such challenges are also faced when considering 2-AG as a pharmacological biomarker for use in future early phase clinical trials with JNJ-69095897. First, 2-AG and AEA, and possibly related endogenous substances arachidonic acid (AA) and prostaglandin E2 (PGE2) levels demonstrate circadian changes in plasma, and might show a similar profile in the cerebrospinal fluid (CSF) but this is currently not known. Second, since CSF is the source of 2-AG most proximal to the intended target of MAGL inhibitors, it is expected to provide the most relevant information on 2-AG as a pharmacological biomarker.
However, obtaining CSF involves a relatively invasive procedure that requires placement of intrathecal catheter at lumbar site and serial CSF sampling[21]. Thus, the most optimal CSF sampling schedule is required to minimize burden to research subjects. Third, is not certain that peripheral 2-AG actually reflects 2-AG in the CSF, and it is unclear whether circadian variation is similarly prominent in the CSF. Fourth, 2-AG is chemically unstable in vitro, and therefore prone to molecular rearrangement to a thermodynamically more stable isomer 1-AG after. Fourth, the intimate relationship between the eCB system and neurovegetative functions such as sleep and stress on the one hand, and homeostasis and neuroplasticity on the other hand, makes it important to interpret 2-AG in the context of neuroendocrine and/or growth factors which form part of the orchestrated circadian oscillation.
Although rigorous laboratory measures such as predefined mealtimes and sleep times are put in place, it remains crucial to characterize hypothalamus-pituitary-adrenal (HPA) axis activity and growth factor activity to adequately validate 2-AG as a pharmacological biomarker for MAGL inhibitors.
In summary, thorough investigation characterizing 2-AG and its possible influencers is warranted before 2-AG can considered a valid
and reliable pharmacological biomarker for MAGL inhibition. The current study is therefore intended to characterize 2-AG in both central and peripheral compartments as a potential pharmacological biomarker in healthy human subjects, to inform thus our future clinical proof of concept trials with the central MAGL inhibitor JNJ-69095897.

• Characterize the plasma and Cerebrospinal Fluid (CSF) time concentration profile of 2-AG, 1-arachidonoylglycero (1-AG) over 24h
and 16h period, respectively.
• Determine the optimal sampling frequency of 2-AG, 1-AG for future studies.
• Establish whether 2-AG and 1-AG in CSF demonstrate a diurnal rhythm.

Day -28(screening) till EOS

N.A.