Experimental strategies to discover and develop the next generation of psychedelics and entactogens as medicines.

Research on classical psychedelics (psilocybin, LSD and DMT) and entactogen, MDMA, has produced a renaissance in the search for more effective drugs to treat psychiatric, neurological and various peripheral disorders. Psychedelics and entactogens act though interaction with 5-HT2A and other serotonergic receptors and/or monoamine reuptake transporters. 5-HT, which serves as a neurotransmitter and hormone, is ubiquitously distributed in the brain and peripheral organs, tissues and cells where it has vasoconstrictor, pro-inflammatory and pro-nociceptive actions. Serotonergic psychedelics and entactogens have known safety and toxicity risks. For these drugs, the risks been extensively researched and empirically assessed through human experience. However, novel drug-candidates require thorough non-clinical testing not only to predict clinical efficacy, but also to address the risks they pose during clinical development and later after approval as prescription medicines. We have defined the challenges researchers will encounter when developing novel serotonergic psychedelics and entactogens. We describe screening techniques to predict clinical efficacy and address the safety/toxicity risks emerging from our knowledge of the existing drugs: 1) An early-stage, non-clinical screening cascade to pharmacologically characterise novel drug-candidates. 2) Models to detect hallucinogenic activity. 3) Models to differentiate hallucinogens from entactogens. 4) Non-clinical preclinical lead optimisation technology (PLOT) screening to select drug-candidates. 5) Modified animal models to evaluate the abuse and dependence risks of novel psychedelics in Safety Pharmacology testing. Our intention has been to design non-clinical screening strategies that will reset the balance between benefits and harms to deliver more effective and safer novel psychedelics for clinical use. This article is part of the Special Issue on 'National Institutes of Health Psilocybin Research Speaker Series'.

In vitro preclinical lead optimisation technologies (PLOTs) in pharmaceutical development.

The explosion of genuine high throughput technologies has allowed large compound libraries to be screened with ever-increasing biological specificity, exacerbating the problem of lead candidate selection for subsequent drug development. To avoid creating a bottleneck, compounds identified from the high throughput screens undergo lead optimisation by employing medium-throughput screen which permits ranking in terms of their basic absorption, distribution, metabolism, excretion (ADME) and toxicological properties. The historical role of the CRO in the drug discovery/development continuum has been to perform efficacy and toxicology studies, simply to support the regulatory submission of lead candidates. This situation is, however, changing with the development of preclinical lead optimisation technologies facilitating the selection of leading candidates, thereby bridging the gap between high throughput efficacy screens and conventional safety assessment programmes.

The neurotoxic effects of methylenedioxymethamphetamine (MDMA) and its metabolites on rat brain spheroids in culture.

Rat whole-brain spheroids were used to assess the intrinsic neurotoxicity of methylenedioxy-methamphetamine (MDMA, Ecstasy) and two of its metabolites, dihydroxymethamphetamine (DHMA) and 6-hydroxy-MDMA (6-OH MDMA). Exposure of brain spheroids to MDMA or the metabolite 6-OH MDMA (up to 500 micromol/L) for 5 days in culture did not alter intracellular levels of glutathione (GSH), glial fibrillary acidic protein (GFAP) or serotonin (5-HT). In contrast, exposure to the metabolite DHMA, which can deplete intracellular thiols, significantly increased GSH levels (up to 170% of control) following exposure to 50 and 100 micromol/L DHMA. There was also a significant reduction in the levels of glial fibrillary acidic protein (GFAP) and GSH by DHMA at the highest concentration tested (500 micromol/L) but there was no effect on 5HT. This may constitute a sublethal neurotoxic compensatory response to DHMA in an attempt to replenish depleted intraneural GSH levels following metabolite exposure. Rat whole-brain spheroids may thus be a useful in vitro model to delineate mechanisms and effects of this class of neurotoxin.

NMDA-induced increases in rat brain glutamine synthetase but not glial fibrillary acidic protein are mediated by free radicals.

Both excitotoxicity and oxidative stress are implicated in the pathophysiology of central nervous system (CNS) ischaemia-reperfusion injury whereby astrocytes offer neural protection through the production of endogenous antioxidants and removal of glutamate from the extracellular milieu. This study investigated whether exogenous alpha-tocopherol, an antioxidant, could prevent N-methyl-D-aspartate (NMDA)-produced increases of the glial specific proteins, glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) in rat brain spheroids in vitro. NMDA (320 microM; 3 days in vitro (DIV)) was unable to induce lipid peroxidation in rat brain spheroids implying that excitotoxicity in this system did not involve substantial free radical formation. However at non-cytotoxic concentrations, increases in astroglial GS were prevented by alpha-tocopherol treatment, suggesting a role for ROS in the excitotoxic process. In contrast, NMDA-induced increases in GFAP remained unchanged by alpha-tocopherol indicating that oxidative stress may not be involved in reactive gliosis at non-cytotoxic NMDA concentrations.

Gliotoxicity in brain reaggregate cultures caused by oxidants and excitatory amino acids can be prevented by alpha-tocopherol and MK-801.

Glutamine synthetase (GS) is a key enzyme involved in glutamate compartmentalisation which may be pivotal in the course of both central free-radical mediated and excitotoxic events. The ability of the oxidants FeCl2 and H2O2 and the excitatory amino acid, N-methyl-D-aspartate (NMDA) to induce changes in astrocytic GS and glial fibrillary acidic protein (GFAP), were assessed in whole rat brain reaggregate cultures. Both FeCl2 and H2O2 reduced GS activity whereas NMDA produced a large increase in enzyme activity. GFAP was not altered significantly by either oxidant although NMDA increased the level of this protein. These effects on such astroglial markers could be reversed in vitro following exposure to a-tocopherol (FeCl2 and H2O2) and MK-801. This study therefore demonstrates that inactivation of GS can be caused by free radical insult whereas stimulation of brain GS and reactive gliosis is produced by excitatory amino acids acting at neuronal NMDA receptors. The study of these gliotoxic events in 3-dimensional reaggregate cultures suggests that this model may be used to detect neuroprotective effects of novel pharmacological agents.

Rat brain mast cells: an in vitro paradigm for assessing the toxic effects of neurotropic therapeutics.

Neurotrophic factors (NTFs) such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) are currently being explored as novel therapeutics in a range of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. To this end, animal studies and clinical trials have been conducted to assess the toxic effects of recombinant NTFs. It is apparent that both NGF and BDNF induce a range of adverse effects, for example inflammation, hyperalgesia, and disturbances in CNS biogenic amine levels which variously manifest as weight loss/gain, changes in feeding behaviour and general malaise. It has been demonstrated that NGF induces release of biologically active mediators, such as histamine, from rat peritoneal mast cells (RPMC). However, whether other NTFs do likewise or indeed are able to induce secretion from other mast cells types had not been explored. We have developed a novel protocol for dispersing mast cells from rat brain tissue, in particular the thalamus which contains the highest number of mast cells in the adult rat. Rat brain mast cells (RBMC) released histamine in a concentration dependent manner in response to NTFs, with a rank order of BDNF > CNTF > NGF; in contrast RPMC were refractory to the effects of BDNF and CNTF. The ability of NTFs to induce release of histamine (a neurotransmitter and neuromodulator in the CNS) from RBMC may go some way to explain some of the adverse effects apparent in vivo upon dosing with NTFs. Mast cells in vitro, and brain mast cells in particular, offer the potential to screen novel NTFs for their neuroimmunotoxic potential relevant to detecting likely clinical adverse effects in humans.

Characterisation of a functional polyamine site on rat mast cells: association with a NMDA receptor macrocomplex.

Polyamines can modulate activation of N-methyl-D-aspartate (NMDA) receptors by binding to a specific polyamine site associated with a NMDA receptor macrocomplex. Polyamines induce histamine release from mast cells, although the mechanism had not been defined. We have examined whether spermine, a natural polyamine, and compound 48/80, regarded as a synthetic polyamine, activate mast cells by a polyamine site associated with a NMDA receptor macrocomplex. Spermine induced secretion of histamine from rat peritoneal mast cells and rat brain mast cells in a concentration-dependent manner. Rat peritoneal mast cells were used as a model system to explore the effects of NMDA antagonists on polyamine-induced histamine release. Ifenprodil, MK801 and arcaine inhibited histamine secretion from mast cells exposed to polyamines; the percentage inhibition was greater against spermine than compound 48/80. These data support the proposal that spermine (and possibly compound 48/80) induce histamine release from mast cells by interacting with a specific polyamine site on a NMDA receptor complex.

Reversal of aluminium-induced metabolic changes in primary rat midbrain neural cultures by the NMDA antagonist MK-801.

Rat embryonic midbrain mixed primary neural cultures were exposed to aluminium chloride at concentrations of 1 x 10(-6) M (low level) or 7.4 x 10(-3) M (high level). Neural cellular metabolic responses as assessed by the neutral red (lysosomal response) and MTT (mitochondrial response) assays indicated that aluminium induced early enhanced neural metabolism at low concentrations in vitro in contrast to depressed metabolism/cellular viability at high concentrations. The excitatory amino acid N-methyl-d-aspartate receptor antagonist MK-801 (5 x 10(-8) M) reversed these metabolic rises at low aluminium levels but not the cell viability loss at higher concentrations. These data are suggestive of an excitotoxic component in the aluminium-induced neural metabolic changes in cultured central nervous systems neural cells, and the possible involvement of oxidative stress.

Mast cells in neuroimmune function: neurotoxicological and neuropharmacological perspectives.

Mast cells are located in close proximity to neurons in the peripheral and central nervous systems, suggesting a functional role in normal and aberrant neurodegenerative states. They also possess many of the features of neurons, in terms of monoaminergic systems, responsiveness to neurotrophins and neuropeptides and the ability to synthesise and release bioactive neurotrophic factors. Mast cells are able to secrete an array of potent mediators which may orchestrate neuroinflammation and affect the integrity of the blood-brain barrier. The 'cross-talk' between mast cells, lymphocytes, neurons and glia constitutes a neuroimmune axis which is implicated in a range of neurodegenerative diseases with an inflammatory and/or autoimmune component, such as multiple sclerosis and Alzheimer's disease. Mast cells appear to make an important contribution to developing, mature and degenerating nervous systems and this should now be recognised when assessing the neurotoxic potential of xenobiotics.

Human placental mast cells as an in vitro model system in aspects of neuro-immunotoxicity testing.

1. In both the developing and adult nervous systems, nerve growth factor (NGF) influences neuronal survival, differentiation and recovery following insult. 2. The effect of NGF upon human placental mast cells (HPMC) was investigated, since it is known that rodent mast cells express a functional receptor for NGF and secrete histamine upon challenge with this neurotrophic factor. Furthermore, human placental tissue contains a significant amount of NGF and expresses a NGF receptor. 3. HPMC were shown to secrete histamine in a concentration dependent manner in response to NGF (0.001-10.0 micrograms ml-1) in the presence of the lipid cofactor phosphatidylserine (10.0 micrograms ml-1). 4. NGF induced histamine release from isolated HPMC with an EC50 of 0.1 microgram ml-1 NGF and maximal secretion of total cellular histamine of 22.3 +/- 3.4% at 3.0 micrograms ml-1. 5. The response was shown to be a secretory process, dependent upon the presence of exogenous calcium ions and to be pH- and temperature-sensitive. 6. HPMC are suggested to be a suitable primary cell model for use in aspects of in vitro toxicity testing, in terms of assessing the neuro-immunotoxic potential of neurotrophic therapeutics. In addition, mechanistic studies concerning those xenobiotics which may exert their neurotoxic effect via interaction with neurotrophic factors and, or their receptors, may be studied in this human cell model.

Modulation of thyroxine uptake and efflux in vitro by temelastine and phenobarbital in cultured hepatocytes from different species, in relation to toxicological effects on the thyroid gland.

Toxicological studies in the rat with phenobarbital and temelastine (SK&F 93944) are associated with thyroid lesions, characterized by thyroid stimulated hormone-mediated thyroid follicular cell hypertrophy and hyperplasia. It has previously been demonstrated that these compounds enhance the hepatocellular accumulation and clearance of thyroxine (T(4)), in rat but not dog or mouse. In this study these events were further characterized in vitro using cultured hepatocytes from different species. Exposure of rat hepatocytes in vitro to phenobarbital and temelastine produced significant increases (P < 0.05) in hepatocellular [(125)I]l-thyroxine accumulation, following 3 hr of exposure to either drug (at a concentration of 10 mum), in the presence of [(125)I]T(4) (0.107 nm final concentration). At this concentration the accumulation of [(125)I]T(4) after xenobiotic exposure was 132.6 +/- 1.5% (phenobarbital) and 135 +/- 2.0% (temelastine) of control values. There was no apparent xenobiotic-induced cytotoxicity (as determined by the mitochondrial MTT index) up to 20 mum temelastine and 50 mum phenobarbital in rat hepatocytes. Experiments performed at 4 degrees C [or under conditions of cellular ATP depletion, induced by 1 mum carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) treatment] failed to show any such increase in hepatocellular thyroxine accumulation. Pretreatment of hepatocytes with either phenobarbital or temelastine for 3 hr before the addition of radiolabelled thyroxine produced increases in hepatocellular hormone accumulation similar to those observed when [(125)I]T(4) and drug were added to the cultures simultaneously. The earliest time at which any increase in [(125)I]T(4) accumulation was observed after drug exposure was approximately 90 min. Exposure of hepatocytes from guinea pig or beagle dog to phenobarbital or temelastine in vitro failed to produce similar increases in hepatocellular [(125)I]T(4) accumulation, demonstrating species specificity of the xenobiotic effect in vitro.

Rodent and human mast cells as an in vitro model in neuroimmunotoxicity testing.

Mast cells derived from rodent or human tissues, either by direct lavage or following enzymic dispersal, secrete biogenic amines on challenge with a range of xenobiotics. In addition to synthetic pathways, re-uptake systems and metabolizing enzymes for histamine and 5-hydroxytryptamine, mast cells are responsive to neurotrophic factors, for example nerve growth factor (NGF). In these studies, rat peritoneal mast cells and human placental mast cells have been compared in terms of histamine release induced by a number of compounds. Of particular interest, it has been demonstrated that these mast cell populations functionally respond to NGF. Since NGF is essential to the development and survival of populations of nerve cells in the peripheral and central nervous systems, potential neurotoxicants or exogenously applied growth factors may interact with this trophic factor and/or its receptor to produce apparent toxicity. It is suggested that mast cells may be a suitable primary cell model for use in aspects of in vitro neurotoxicity and neuroimmunotoxicity testing for xenobiotics interfering with NGF function, for investigating the effects of neurotoxicants on monoamine function and for studying mechanistic aspects of neurodegenerative diseases.

Phase 1 of an in vitro neurotoxicological pre-validation trial.

In the absence of a formal in vitro alternative model for neurotoxicity testing, the CellTox Centre, in collaboration with the University of Salford, FRAME and the EEC, has set up an initial pre-validation trial of a three-tiered scheme that was originally proposed in 1989. Using a cell battery of neuroblastomas and primary neural cultures (Tier I) and primary astrocytes (Tier III) an initial set of 20 chemicals (out of a reference set of 40 chemicals) has been assessed for neurotoxicity using a multiple end point assay system. The methods used were relatively simple, rapid and inexpensive, and the chemical end points were easily quantified. The results of the pre-validation trial have enabled us to revise the first tier of the proposed tiered system for full micro-validation.

Use of astrocytes for in vitro neurotoxicity testing.

The use of primary cultures of astrocytes as indicators of toxic potential was assessed using 20 selected compounds. Multiple endpoints were used to evaluate astrocyte reactions. Trypan blue dye exclusion and total cellular protein content of the cells were used as general indices. EC(50) values from the trypan blue experiments could be used to rank toxicity of compounds in a manner that correlates well with known toxicity for compounds that have specific astrocyte toxicities. Neurone specific neurotoxicants had no measurable effects on astrocytes indicating that this system differentiates gliotoxicity from neurotoxicity. Protein content, and content of the astrocyte specific glial fibrillary acidic protein (GFAp), were seen to increase at lower doses of gliotoxic compounds. This phenomenon appears to be similar to reactive gliosis in vivo, as assessed by immunostaining, and is an extremely sensitive indication of cellular damage. Support studies using astrocyte uptake of 2-deoxy glucose showed a similar pattern of activation in the cells as protein increases. This has been confirmed using the nonisotopic technique of MTT reduction.

Effects of the neurotoxin ethylcholine mustard aziridinium in rat brain reaggregate cultures.

The toxicity was studied of ethylcholine mustard aziridinium (ECMA) in rat brain reaggregate cultures. The objective was to define optimum conditions for selective effects on cholinergic neurones. Single treatments with 12.5-50 mum-ECMA caused approximately 35% loss of choline acetyltransferase (ChAT) activity in 2 hr, increasing to 70-80% in 72-120 hr. The 2-hr, but not the longer-term loss of activity was prevented by the choline transport blocker hemicholinium-3 (40 mum). Significant loss of ChAT activity could not be obtained with less than 12.5 mum-ECMA. Approximately 55% irreversible inhibition of muscarinic receptor binding also occurred in 2 hr. However, after 12.5 mum-ECMA, binding recovered to control values within 48 hr. These persisted throughout the longer-term more extensive loss of ChAT activity, which suggests that receptor recovery localized to cells other than cholinergic neurones. After 25-50 mum-ECMA, muscarinic receptor binding did not recover, which suggests more widespread cytotoxicity destroying cells other than the cholinergic neurones. More pronounced leakage of lactate dehydrogenase and reduced reaggregate concentrations of total and neurofilament protein were consistent with more generalized cytotoxicity after 25 or 50 mum-ECMA. Examination of ECMA treated reaggregates or cerebellar monolayer cultures by light microscopy confirmed this. Concentrations of 12.5 mum-ECMA therefore represent the optimum for achieving selective toxic effects on cholinergic neurones in the cultures. After ECMA, reaggregate concentrations of 5-hydroxyindole acetic acid were markedly increased (100-300%), which suggests increased activity of 5HT neurones. This demonstrates that reaggregate cultures might be used to study trans-synaptic neurochemical sequelae of brain cholinergic neurone loss.

Aluminium and iron induced metabolic changes in neuroblastoma cell lines and rat primary neural cultures.

The acute effects of low concentrations of aluminium (10(-12)-10(-3)m) in neural cell culture were investigated over 1-48 hr. Mitochondrial and lysosomal activities were used as measures of metabolic change after aluminium administration to C1300 mouse-derived neuroblastoma cells (C1300 N2A) and primary mixed cultures of rat embryonic mid-brain. Very rapid increases in mitochondrial and lysosomal activity occurred over periods as short as 3 hr. The aluminium-induced metabolic changes in C1300 N2A cells were compared with those produced by iron. The similar time course of metabolic events observed with iron is suggestive of a similar neurotoxicological mechanism. Experiments examining the effect of preincubation with the free radical scavenger, alpha-tocopherol (200 mum), on the aluminium and iron induced changes in primary rat mid-brain cultures, showed an inhibition of these short-term metabolic events.

New models for the In vitro assessment of neurotoxicity in the nervous system and the preliminary validation stages of a 'tiered-test' model.

Many cell culture models are available for the in vitro assessment of neurotoxicity. The use of three culture types has been investigated: neuroblastoma cell lines, primary cultures of rat and chick midbrain, and organotypic whole brain reaggregate cultures. A tiered system has been proposed involving hierarchical testing through three layers of different neural complexities. This scheme is currently undergoing validation under the auspices of FRAME/EC using 40 test chemicals. To determine the performance and suitability of these culture models studies on selected neurotoxins have been performed: ethylcholine mustard aziridinium, vincristine, aluminium, glutamate, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and T(3)-deprivation. Aspects of this work are described, including mechanistic investigations in rat brain reaggregate cultures. In vitro exposure of xenobiotics through a tiered testing system (ranging from simple cell-based assays measuring cytotoxicological parameters to more complex markers in organotypic cultures) may permit detection of central nervous system neurotoxicity in the contexts of both 'screening' and mechanistics. The degree of simplicity, automaticity and transportability of the tests requires consideration as will the possibility of endpoints for specific classes of chemicals, for example cholinesterase for organophosphorus insecticides. Factors such as extrapolation from the central nervous system to the peripheral nervous system, metabolic activation, the blood-brain barrier, degree of neural cell activation, repair mechanisms, and developing versus adult nervous systems are considered.