Incilius alvarius Cell-Based Synthesis of 5-Methoxy-N,N-Dimethyltryptamine

Abstract

There is growing interest in the therapeutic potential of 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) for psychiatric disorders. Although 5-MeO-DMT can be chemically synthesized, the parotoid gland secretions of Incilius alvarius (also known as the Colorado River or Sonoran Desert toad) contain 5-MeO-DMT and other molecules including bufotenine, bufagenins, bufotoxins, and indole alkylamines that may have individual clinical utility or act as entourage molecules to enhance the activity of 5-MeO-DMT. I. alvarius is currently under severe ecological pressure due to demand for natural 5-MeO-DMT and habitat loss. We established a cell line from tissue obtained by wedge biopsy of the I. alvarius parotoid gland and confirmed the cell-based biosynthesis of 5-MeO-DMT by liquid chromatography with tandem mass spectrometry. Cell-based biosynthesis of I. alvarius parotoid gland secretions is a potentially cruelty-free and sustainable source of naturally derived 5-MeO-DMT for research and drug development.

Introduction

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is the primary psychoactive component of the parotoid gland secretion (“venom”) of Incilius alvarius, the Sonoran Desert or Colorado River toad, and accounts for 15% of the dry weight of the secretion.¹ 5-MeO-DMT primarily acts as an agonist at the 5-HT_1A and 5-HT_2A receptors with a higher affinity for the 5-HT_1A subtype.² 5-MeO-DMT is O-demethylated by polymorphic cytochrome P450 2D6 (CYP2D6) to an active metabolite, bufotenine.^3–5

Neuropsychiatric disorders including mood and anxiety disorders are leading causes of disability worldwide and place an enormous economic burden on society.^6–8 Serotonergic psychedelics are receiving increasing attention as novel therapeutics for depression and other psychiatric and neurological disorders.⁹ Preclinical and clinical evidence support neuroplasticity as the convergent downstream mechanism of action of psychedelics. Through their serotonin receptor targets, psychedelics including psilocybin, lysergic acid diethylamide, 5-MeO-DMT, and N,N-dimethyltryptamine induce synaptic, structural, and functional changes, particularly in the pyramidal neurons of the prefrontal cortex.¹⁰

5-MeO-DMT appears to be pharmacodynamically unique as compared with other psychedelics in terms of the intensity and rapid onset of action, short duration of effect, and transcriptomic and other parameters.¹¹ An optimized chemical method for the synthesis of 5-MeO-DMT has been described.¹² Volunteers consuming toad secretion containing 5-MeO-DMT experienced a 20–30% increase in the magnitude of subjective effects including “ego dissolution” and “altered states of consciousness” compared with the effects reported by volunteers who used synthetic 5-MeO-DMT.^1,13,14

Although these observations are largely anecdotical and could be attributed to dose, setting, and recall bias, it is also possible that the nonpsychedelic molecules in the toad secretion, also known as the entourage molecules, may be responsible for the perceived enhanced effect of toad-derived 5-MeO-DMT.¹⁵ Whether or how the entourage molecules interact with the psychedelic molecules, thereby modulating the psychoactive experience and the neuroplastic effect, is currently unknown.^1,13,14 Although 5-MeO-DMT is present in several entheogenic plants, it should be noted that due to the high concentrations in the parotoid secretions of I. alvarius, the Sonoran Desert toad is currently under severe ecological pressure due to demand for recreational, self-medication, and spiritual use.¹⁶

Methods

Parotoid gland biopsy

Two I. alvarius toads were donated by an amateur herpetologist. The species was confirmed by an amphibian veterinarian. Parotoid anatomy and morphology were established according to O ´Donohoe et al.¹⁷ A partial parotoid gland biopsy was undertaken by an amphibian veterinary surgeon under aseptic conditions on the two anesthetized I. Alvarius toads in conformity with the current Institutional Animal Care and Use Committee (IACUC) best practice for the handling of laboratory animals. In total, 500 mL deionized water, half a tablespoon of NaHCO₃ (Sigma-Aldrich, St Louis, MO), and half a tablespoon of MS222 (Sigma-Aldrich) (1:1 ratio) were mixed to make a ∼7.38 pH buffered anesthetic solution (temperature = 21.6°C).

After 10 to 15 min, the plane of anesthesia was reached. A slightly modified procedure as described by Simoncelli et al¹⁸ was performed; full-thickness ventral skin was removed, and a 4 to 6 mm punch biopsy was taken from either the left or the right parotoid gland. The wound was closed with 4–0 Maxon™ suture (Medtronic, Minneapolis, MN) and surgical glue. The toads were kept in a warm water bath with oxygen administration until reflexes recovered. Sutures were removed after 14 days.

Explants were then rinsed with 70% isopropanol and phosphate-buffered saline, and stored in an ultracold freezer until culture initiation. Toad care followed generally accepted IACUC protocols for the best practice in the handling of laboratory animals and the toads remain alive and thriving to date, 2 years after the procedure. They live in a 90 × 43 × 44 cm heat- and humidity-controlled terrarium next to a window receiving natural light. During winter, timed light is provided to ensure 12 h of light.

Their terrarium contains a habitat hut and two water bowls that are refilled with treated water. The coconut bedding is changed every 2 weeks. The toads are fed every second day vitamin dust enriched crickets and worms. A veterinarian technician inspected and weighed the toads weekly after the operation for 3 months and monthly since.

Cell culture, immortalization, and maintenance

Medium preparation described by Ellinger et al⁵ was employed with modification and composed of 2 × L-15 (GIBCO, Thermo Fisher Scientific, Waltham, MA), 10% fetal bovine serum (Sigma Aldrich), and a 1% streptomycin, amphotericin, and fungizone (Sigma Aldrich) solution.

Two treatment groups were established: gelatin (Sigma-Aldrich) was applied to two 24-well plates, whereas no adherent was applied to two 24-well plates. Explants were subsequently thawed in a 65°C water bath, rinsed with amphibian Ringers solution prepared as described by Handler et al,¹⁹ resuspended in fresh medium, and prepared for connective tissue digestion. Two treatments of cell isolators were used: collagenase was employed similarly to Okumoto et al²⁰ followed by enzyme-free cell dissociation solution.

Upon completion, dissociated cells were strained via both 0.40 and 0.22 μm cell strainers. Isolated cells were aseptically seeded into 24-well plates and incubated at 25°C and 5% CO₂. Upon confluence, cells were disassociated with the initial reagent used and passaged to additional well plates. Medium exchange occurred approximately every 3–4 days and cell immortalization was achieved using the SV40 T Antigen Cell Immortalization Kit (Alstem, Richmond, CA). Cell culture was undertaken in an incubator at 25°C and 5% CO₂.

Upon confluence, the cells were disassociated and passaged with medium exchange every 3–4 days. Medium was obtained when cell medium change was required; cells were in medium for 3–4 days before it was removed for analysis. The volume removed with an aseptic syringe was ∼1–2 mL per well.

The culture was maintained for 45 days as this experiment was a feasibility study limited in time. The remaining cells were transferred to a research partner for further analysis.

Liquid chromatography–mass spectrometry analysis

Before analysis, cell medium was filtered using an Amicon Ultra-0.5 Centrifugal Filter Unit (molecular weight cut-off 10 kDa, Millipore Sigma, Burlington, MA). 5-MeO-DMT was quantified using an UPLC—Xevo TQ-S micro MS system (Waters Corporation, Milford, MA). The calibration curve (Supplementary Fig. S1) was prepared using the 5-MeO-DMT analytical standard, in methanol, from Cerilliant Corporation (Round Rock, Texas) using a concentration range of 1–2000 ng/mL. For quantification, monitored ions at 130.25 and 159.07 m/z were used.

The column used was a Phenomenex (Torrance, CA) Luna Omega 3 μm Polar C18 (150 × 4.6 mm) at 40°C, solvent A was water and 0.1% formic acid and solvent B was acetonitrile and 0.1% formic acid at a flow rate of 0.6 mL/min. The injection volume is 1 μL. The mass spectrometry (MS) was used in positive ion mode with time-scheduled multiple reaction monitoring (MRM) acquisition. The source temperature was 150°C, capillary voltage 1.00 kV, desolvation temperature 600°C, desolvation gas flow of 1000 L/h, with a cone gas flow of 75 L/h.

In the positive-ion mode and under turbo-ion-spray ionization conditions, 5-MeO-DMT gave a precursor ion (M+H)+of m/z 219.20. Product ion of m/z 159.07 and 130.25 was found to be predominant for 5-MeO-DMT under the collision energy of 40 V (the MRM method was developed by utilizing IntelliStart Software, Waters Corporation). The MRM transitions m/z 219.2 → 159.07 and 219.2 → 130.25 were chosen to analyze 5-MeO-DMT, which offered the strongest signal compared with other MRM transitions. UV tracers for parotoid secretion, water +1 ng/mL 5-MeO-DMT, and parotoid cell-free medium are provided in Supplementary Figure S2.

Results and Discussion

Parotoid cells obtained from biopsies of anesthetized I. alvarius toads were successfully immortalized. Cultures were maintained for 45 days. Dried medium from the cell culture had a light tan appearance that was similar to the known appearance of dried I. alvarius parotoid secretion. Well plate medium samples were collected at 12 and 36 days from initiation and were analyzed for the presence of 5-MeO-DMT using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and compared with spontaneously secreted material from an I. alvarius toad. MS fragmentation of the medium samples and secreted material showed conformity in structure with 5-MeO-DMT (Fig. 1). The signal-to-noise ratio in the parotoid secretion was much higher than in the cell culture medium.

Fig. 1.

Chromatograms of 1 μL injection of (i) parotoid secretion (ii) parotoid cell-free medium, and (iii) blank medium +1 ng/mL 5-MeO-DMT. (A) Full scan MS, total ion chromatogram. (B) Sum of MRM transitions of 219.20 → 159.07 and 219.20 → 130.25. (C) Identification and quantification of MRM transitions of (i) 219.20 → 159.07 and (ii) 219.20 → 130.25 from parotoid cell-free medium. MRM, multiple reaction monitoring.

Additional chromatographic peaks were present but were not identified due to the paucity of the material.

In this pilot study, small quantities were produced, and it was not possible to accurately determine concentration or yield. We are conducting research to demonstrate scalable production of 5-MeO-DMT in the parotoid cell line and further confirm de novo biosynthesis. This study demonstrates the potential availability of naturally derived 5-MeO-DMT produced through cell culture, as opposed to the cruel and destructive practice of “milking” I. Alvarius, and supports efforts to ensure the protection of our planet's entheogen heritage. An independent review board comprising of a veterinarian, laboratory scientist and a member of the public (with an interest in animal welfare) supervised the research.

Authors' Contributions

The article was written with the contributions of all authors. All authors have approved the final version of the article.

Author Disclosure Statement

L.L. holds an equity in Back of the Yards Algae Sciences.

Funding Information

No funding was received for this article.