Overcoming the clinical challenges of traditional ayahuasca: a first-in-human trial exploring novel routes of administration of N,N-Dimethyltryptamine and harmine

Recently, the Amazonian plant medicine “ayahuasca”—containing the psychedelic compound N,N-dimethyltryptamine (DMT) and numerous β-carboline alkaloids, such as harmine—has been suggested to exhibit beneficial effects in patients with affective and other mental health disorders. Although ayahuasca ingestion is considered safe, its pharmacokinetics/pharmacodynamics and tolerability profile pose some challenges and may limit the clinical applicability in vulnerable patient populations. While overdosing and the admixture of intolerable plant constituents may explain some of the common adverse reactions, the peroral route of administration may represent another relevant source of gastro-intestinal intolerabilities and unpredictable pharmacokinetics across users. To overcome these challenges, the present work aimed at creating ayahuasca-analogue formulations with improved pharmacokinetics and tolerability profiles. To this end, we developed peroral formulas and compared them with parenteral formulas specifically designed to circumvent the gastro-intestinal tract. In more detail, peroral administration of a capsule (containing purified DMT and harmine) was tested against a combined administration of an oromucosal harmine tablet and an intranasal DMT spray at two dose levels in an open-label within-subject study in 10 healthy male subjects. Pharmacokinetic and pharmacodynamic profiles were assessed by means of continuous blood sampling, vital sign monitoring, and psychometric assessments. Common side effects induced by traditional herbal ayahuasca such as nausea, vomiting, and diarrhea were significantly attenuated by our DMT/harmine formulations. While all preparations were well tolerated, the combined buccal/intranasal administration of harmine and DMT yielded substantially improved pharmacokinetic profiles, indicated by significantly reduced variations in systemic exposure. In conclusion, the combined buccal/intranasal administration of harmine and DMT is an innovative approach that may pave the way towards a safe, rapid-acting, and patient-oriented administration of DMT/harmine for the treatment of affective disorders.Clinical Trial Registration:clinicaltrials.gov, identifier NCT0471633

Conformational dynamics of cationic cys-loop receptors

Understanding the functional dynamics of ligand-gated ion-channels (LGIC's) on the molecular level is the aim of this thesis. Members of the protein family of LGIC's are involved in the fast synaptic signal transmission and thereby play a central role in inter-cellular communication in higher multi-cellular organisms. Here, two prototypical members of ligand-gated cys-loop receptors were studied: the homopentameric type 3 serotonin receptor (5HT3AR) and the muscle-type nicotinic acetylcholine receptor (nAchR). The single-channel kinetics of the 5HT3AR was investigated using single-channel patch-clamp and single-molecule fluorescence techniques. Our principal findings are that the 5HT3AR channel shows up to five, agonist activated, nearly equidistant conductance levels, by binding of up to five agonist molecules. Detailed analysis of data obtained using serotonin the native agonist and quipazine-TMR, a fluorescent partial agonist at different ionic conditions has been performed. An allosteric calcium modulation was observed with serotonin but not with the partial agonist indicating receptor activation with altered conformational pathways. In conclusion, a functional model is proposed, describing a two-way conformational activation. Binding of each agonist opens a "conductance filter" within the addressed subunit. Additionally, a global gate, which can be modulated by the presence of calcium ions, determines the channel kinetics. In order to identify at least one of the hypothesized conformational pathways of the 5HT3A receptor activation, a mutation of aspartate to cysteine at the position 298 was introduced into the receptor variant devoid of cysteine residues. Labelling the mutated residue with a fluorescent dye resulted in a fluorescence quenching upon receptor activation. This effect was interpreted as a functional conformational rearrangement. Although no specific time-scale of such a (local or global) change in conformation could be determined on the molecular level, the application of the described approach as a possible test for functionality upon receptor solubilisation is discussed. The simultaneous observation of the discrete ligand binding and the subsequent single-channel gating was discussed for years in the LGIC field as the method of choice for direct and unbiased examination of the channel activation process. Here, current recordings of single-channel gating events were combined with optical single-molecule detection of fluorescent agonist binding. The time delay Δt between ligand binding and gating events of a gain-of-function variant of the foetal and the wild-type adult nAchR were measured on the single molecule level on living cells. For times above approximately 1 ms, the distribution of Δt for both receptor types obeys a power-law with an exponent close to 3/2, corresponding to a normal conformational diffusion. Increasing the time resolution, a monoexponential function describes the obtained data best for times below 1 ms, suggesting that an activation barrier in the free energy landscape of the receptor activation can be overcome within one step. Finally, a novel method for the fast and simple induction of synapse formation was developed. Optical tweezers were used for pulling membrane-nanotubes out of the plasma membrane of a living cell. Connecting these nanotubes to the cell membrane of up to 100 µm distant cells, axonal or dendritic protrusions can be mimicked. Functional electrical synapses, based on gap junctions, were artificially created. The results obtained in this work contribute to the functional characterisation of LGIC's in living cells, yielding models that might be extrapolated as concepts of conformational dynamics of proteins in general

Membrane nanotubes drawn by optical tweezers transmit electrical signals between mammalian cells over long distances

Biological cells continuously change shape allowing essential functions such as cell motility, vesicle-mediated release/uptake of soluble and membrane components or nanotube-mediated cell–cell communications. Here we use single cell micromanipulation to induce functional changes of cell shape for nanobiotechnological applications. Optical tweezers are focused on the plasma membrane of living cells to pull membrane nanotubes of similar200 nanometre diameters and 100 micrometre lengths. Upon switching off the laser tweezer membrane nanotubes relax back to the cell surface. Single-exponential relaxation times deliver local mechanical properties of cells' plasma membrane. Nanotubes pulled beyond 100 micrometre tear off and form micrometre-sized vesicles carrying functional membrane receptors and cytoplasmic signaling components. Membrane nanotubes from one cell can be contacted to adjacent cells forming via connexins intercellular electrical connections within seconds in all directions. Our method opens broad applications for multiplexing single-cell analytics to submicrometer/subfemtoliter ranges and for creating artificial intercellular signaling networks, both not attainable by current methodologies

Overcoming the clinical challenges of traditional ayahuasca: a first-in-human trial exploring novel routes of administration of N,N-Dimethyltryptamine and harmine

Recently, the Amazonian plant medicine "ayahuasca"-containing the psychedelic compound N,N-dimethyltryptamine (DMT) and numerous beta-carboline alkaloids, such as harmine-has been suggested to exhibit beneficial effects in patients with affective and other mental health disorders. Although ayahuasca ingestion is considered safe, its pharmacokinetics/pharmacodynamics and tolerability profile pose some challenges and may limit the clinical applicability in vulnerable patient populations. While overdosing and the admixture of intolerable plant constituents may explain some of the common adverse reactions, the peroral route of administration may represent another relevant source of gastro-intestinal intolerabilities and unpredictable pharmacokinetics across users. To overcome these challenges, the present work aimed at creating ayahuasca-analogue formulations with improved pharmacokinetics and tolerability profiles. To this end, we developed peroral formulas and compared them with parenteral formulas specifically designed to circumvent the gastro-intestinal tract. In more detail, peroral administration of a capsule (containing purified DMT and harmine) was tested against a combined administration of an oromucosal harmine tablet and an intranasal DMT spray at two dose levels in an open-label within-subject study in 10 healthy male subjects. Pharmacokinetic and pharmacodynamic profiles were assessed by means of continuous blood sampling, vital sign monitoring, and psychometric assessments. Common side effects induced by traditional herbal ayahuasca such as nausea, vomiting, and diarrhea were significantly attenuated by our DMT/harmine formulations. While all preparations were well tolerated, the combined buccal/intranasal administration of harmine and DMT yielded substantially improved pharmacokinetic profiles, indicated by significantly reduced variations in systemic exposure. In conclusion, the combined buccal/intranasal administration of harmine and DMT is an innovative approach that may pave the way towards a safe, rapid-acting, and patient-oriented administration of DMT/harmine for the treatment of affective disorders.Clinical Trial Registration: clinicaltrials.gov, identifier NCT04716335ISSN:1663-981