Cytochrome P450 (IC50 = 19 μM)

N, N-dimethyltryptamine (DMT) is a psychedelic compound that has shown potential in the treatment of depression. Aside from the primary role of monoamine oxidase A (MAO-A) in DMT metabolism, the metabolic pathways are poorly understood. Increasing this understanding is an essential aspect of ensuring safe and efficacious use of DMT.This work aimed to investigate the cytochrome 450 (CYP) mediated metabolism of DMT by incubating DMT with recombinant human CYP enzymes and human liver microsomes (HLM) followed by analysis using high-resolution mass spectrometry for metabolite identification.DMT was rapidly metabolised by CYP2D6, while stable with all other investigated CYP enzymes. The metabolism of DMT in HLM was reduced after inclusion of harmine and Proadifen/SKF-525A whereas quinidine did not affect the metabolic rate, likely due to MAO-A residues present in HLM. Analysis of the CYP2D6 incubates showed formation of mono-, di- and tri-oxygenated metabolites, likely as a result of hydroxylation on the indole core.More research is needed to investigate the role of this metabolic pathway in vivo and any pharmacological activity of the proposed metabolites. Our findings may impact on safety issues following intake of ayahuasca in slow CYP2D6 metabolizers or with concomitant use of CYP2D6 inhibitors.[2]

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Oxidation of the experimental anti-tumour agent N-[(2'-dimethylamino)ethyl]acridine-4-carboxamide (AC; NSC 601316; acridine carboxamide) to the 9(10H)acridone, followed by ring hydroxylation and glucuronidation, appears to be the main pathway of detoxication of AC in the rat and mouse. The acridone formation has been further characterized in vitro using an enzyme-enriched fraction where activity per milligram protein is increased approximately 10-fold compared with the cytosolic fraction. Inhibition by amsacrine [4'-(9-acridinylamino)methanesulphon-m-anisidide; NSC 249992] and menadione (50% inhibition at 6.4 and 1.8 microM, respectively) but not allopurinol (to 30 microM) indicates that the activity is due to aldehyde oxidase, without the involvement of xanthine oxidase. Interestingly, acridone formation in both the cytosolic and enzyme-enriched fractions is highly sensitive to the classical cytochrome P450 inhibitor Proadifen/SKF-525A [proadifen hydrochloride; 2'-(diethylamino)ethyl 2,2-diphenylpentenoate] (50% inhibition at 9.2 and 1.9 microM, respectively). Further analysis indicates mixed non-competitive type inhibition by SKF-525A (K(is), 0.3 microM; K(ii), 4.9 microM). Little or no inhibition was seen with cimetidine, metyrapone or methimazole. No NADPH-dependent acridone formation was observed with the microsomal fraction. These data indicate that acridone formation previously observed in isolated rat hepatocytes and in vivo is most likely due to aldehyde oxidase rather than cytochrome P450 [3].

The aim of this study was to investigate the effect an inhibitor of cytochrome-P450, Proadifen hydrochloride (SKF525), on the excitability of serotonin neurons. Adult male Wistar rats were administered SKF525 forty-eight, twenty-four, and one hour before electrophysiological assessments. Control animals were injected saline. Rats were anesthetized with chloral hydrate and glass electrodes were stereotaxically inserted into the dorsal raphe nucleus (DRN). Serotonin neurons were identified and their firing activity was recorded. It was found that the SKF525 inhibits the excitability of 5-HT neurons. We suggest that corticosterone might play a key role in the SKF525-induced inhibition of 5-HT neurons.[1]

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The aim of this study was to investigate the effect of Proadifen/SKF525 on the excitability of central catecholamine neurons. Adult male Wistar rats were administered SKF525 forty-eight, twenty-four, and one hour before electrophysiological assessments. Control animals were injected saline. Rats were anesthetized with chloral hydrate and glass electrodes were inserted into the locus coeruleus (LC) or ventral tegmental area (VTA). Noradrenaline neurons of the LC and dopamine of the VTA neurons were identified, and their firing activity was recorded. It was found that the SKF525 enhanced the excitability of noradrenaline and reduced the excitability of dopamine neurons. We suggest that corticosterone-induced inhibition of 5-HT neurons underlines, at least in part, the ability of SKF525 to stimulate noradrenaline neurons. The inhibitory effect of SKF525 on dopamine neurons might be in turn secondary to the stimulatory effect of this compound on noradrenaline neurons [4].

Inhibition of CYP enzymes in HLM [2]
To further assess the contribution of specific CYP enzymes to DMT metabolism, DMT (1 µM) was incubated with HLM (0.5 mg/mL) and an NADPH regenerating system (1.3 mM NADP + 3.3 mM Glucose-6-phosphatase + 3.3 mM magnesium chloride, 0.4 U/mL glucose-6-phosphate dehydrogenase) in phosphate buffer (100 mM, pH 7.4) in the presence or absence of quinidine (a CYP2D6 inhibitor), harmine (inhibitor of MAO-A and CYP2D6) or Proadifen/SKF-525A (a general CYP inhibitor) at a total volume of 1 mL. Samples were pre-incubated at 37 °C for 5 min before the reaction was initiated by addition of HLM. Incubations proceeded at 37 °C for 60 min and aliquots of 100 µL were removed at 0 (before the reaction was initiated), 5, 10, 20, 30 and 60 min. Reactions were quenched by addition of ice-cold acetonitrile (300 µL) spiked with the internal standards DMT-D6 (100 nM) and 2-Me-IAA (300 nM). Samples were centrifuged at 10.000 × g for 2 min, supernatants were transferred into new vials and were either analysed directly or stored at −80 °C before analysis. Experiments were performed in duplicates. DMT half-life was calculated in the same way as for the incubations with recombinant CYP enzymes.

Adult male Wistar rats (200–250 g) were housed in a temperature-controlled room (22–24°C) with a 12:12 hours light-dark cycle, and had ad libitum access to food and water. Rats were allowed to acclimatize for one week after their arrival in our animal facility. Proadifen/SKF525 was dissolved in saline. To achieve the steady-state inhibition of the CYP, the rats received three intraperitoneal (i.p.) injections of SKF525 (25 mg/kg): forty-eight, twenty-four, and one hour before electrophysiological assessments. Control animals were injected saline using the same protocol.[1]

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One hour after the last saline or Proadifen/SKF525 injection, rats were anesthetized with chloral hydrate (0.4 g/kg, i.p.) and mounted into the stereotaxic frame. Rat body temperature was maintained at 37°C with a heating pad. The scalp was opened and a 3 mm hole was drilled in the skull for insertion of electrodes. Glass-pipettes were pulled with a DMZUniversal Puller to a fine tip approximately 1 μm in diameter and filled with 2 M NaCl solution. Electrode impedance ranged from 7 to 8 MΩ. The pipettes were lowered into the DRN, 7.8–8.3 mm posterior to bregma and 4.5–7.0 mm ventral to brain surface (Paxinos and Watson 2014), by a hydraulic micro-positioner. Serotonin neurons were identified by their regular, lowfrequency (less than 5 Hz) firing rate and positive bi- or tri-phasic action potential of the total duration of 2.0–5.0 ms and cumulative duration of depolarization and repolarization phases of 0.8–1.2 ms, as described in the previous studies and recorded for at least two minutes using the Power Lab data acquisition system and Lab Chart software.[1]

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We found a significant (p = 0.03, two-tailed Student’s t-test) 18%-decrease in 5-HT neuronal firing activity in Proadifen/SKF525-administered rats (1.75 ± 0.12 Hz, 119 cells from 7 rats) in comparison to controls (2.14 ± 0.14 Hz, 97 neurons from 8 rats; Fig. 1). The mean number of the spontaneously active 5-HT neurons per electrode track was not statistically different between the groups (SKF525: 5.67 ± 0.95; control: 3.69 ± 0.57; p = 0.08, two-tailed Student’s t-test). As a potent CYP inhibitor, SKF525 was previously reported to increase the plasma levels of corticosterone in rats (Magus et al. 1968). On the other side, corticosterone inhibits the excitatory glutamatergic input to 5-HT neurons of the DRN (Wang et al. 2012). It is therefore possible that corticosterone mediates, at least in part, the inhibitory effect of SKF525 on brain 5-HT neurons. It was previously reported that the suppression of 5-HT neurons by intra-DRN injection of γ-aminobutyric acid (GABA) induced depression-like behavior in mice (Xiao et al. 2017). It is possible that the partial inhibition of 5-HT neurons by SKF525 have a depressogenic effect as well. It was indeed reported that SKF525 reversed the antidepressantlike behavioral effect of imipramine and desipramine in rats (Maj et al. 1981).[1]

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SKF525/Proadifen administration [4]
SKF525 was administrated using the protocol explained in our previous study (Grinchii et al. 2018). SKF525/Proadifen was dissolved in saline. To achieve the steady-state inhibition of the CYP, the rats received three intraperitoneal (i.p.) injections of SKF525 (25 mg/kg): forty-eight, twenty-four, and one hour before electrophysiological assessments. Control animals were injected saline using the same protocol.

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Electrophysiological assessments [4]
The assessment of excitability of catecholamine-secreting neurons was performed as explained in our previous studies (Dremencov et al. 2017; Koprdova et al. 2019; Csatlosova et al. 2021). One hour after the last saline or Proadifen/SKF525 injection, rats were anesthetized with chloral hydrate (0.4 g/kg, i.p.) and mounted into the stereotaxic frame. Rat body temperature was maintained at 37°C with a heating pad. The scalp was opened, and a 3 mm hole was drilled in the skull for insertion of electrodes. Glass-pipettes were pulled with a DMZ-Universal Puller to a fine tip approximately 1 μm in diameter and filled with 2 M NaCl solution. Electrode impedance ranged from 4 to 6 MΩ. The pipettes were inserted into the LC (8.0–8.3 mm posterior to bregma, 1.2–1.4 mm lateral to the midline, and 5.5–7.5 mm ventral to the brain surface) or VTA (4.5–5.5 mm posterior to bregma, 0.6–0.8 mm lateral to the midline, and 7.0–8.5 mm ventral to the brain surface) (Paxinos and Watson 2014) by hydraulic micro-positioner. The signal from the electrodes was amplified ×1000 using the DP-311 Differential Amplifier, filtered of the low-frequency (~50/60 Hz) harmonic with the VDL215EQ2 Graphic Equalizer and Hum Bag Noise Eliminator and fed to the Lenovo B50-35 PC using the Power Lab 4/35 Data Acquisition System with the sampling rate of 100 kHz. The bin size was set at 1 ms. The action potentials generated by monoamine-secreting neurons were recorded using the AD Instruments Extracellular Recording System. Noradrenergic neurons were recognized by action potentials with a long-duration rising phase, regular firing rate of 0.5–5.0 Hz, and a characteristic burst discharge in response to nociceptive pinch of the contralateral hind paw (Vandermaelen and Aghajanian 1983). Dopamine neurons were recognized by tri-phasic action potentials lasting between 3 and 5 ms with a rising phase lasting over 1.1 ms, inflection or “notch” during the rising phase, marked negative deflection, irregular firing-rate of 0.5–10 Hz, mixed single-spike and burst firing with characteristic decrease of the action potentials amplitude within the bursts (Grace and Bunney 1983). The same number of electrode descents per brain structure (four for the LC and five for the VTA) were made in saline- and Proadifen/SKF525-treated rats. All neurons in all groups of animals were recorded for two minutes.

65341 rat LD50 oral 2140 mg/kg Journal of Pharmacology and Experimental Therapeutics., 111(131), 1954 [PMID:13175094]
65341 rat LD50 intraperitoneal 163 mg/kg Psychotropic Drugs and Related Compounds, 2nd ed., Usdin, E., and D.H. Efron, Washington, DC, 1972, -(366), 1972
65341 mouse LD50 oral 538 mg/kg Journal of Pharmacology and Experimental Therapeutics., 111(131), 1954 [PMID:13175094]
65341 mouse LD50 intraperitoneal 110 mg/kg Pharmaceutical Chemistry Journal, 3(508), 1969
65341 mouse LD50 intravenous 60 mg/kg Psychotropic Drugs and Related Compounds, 2nd ed., Usdin, E., and D.H. Efron, Washington, DC, 1972, -(366), 1972

[1]. Inhibition of cytochrome P450 by proadifen diminishes the excitability of brain serotonin neurons in rats. Gen Physiol Biophys. 2018 Sep;37(6):711-713.

[2]. N, N-dimethyltryptamine forms oxygenated metabolites via CYP2D6 - an in vitro investigation. Xenobiotica. 2023 Dec;53(8-9):515-522.

[3]. Inhibition by SKF-525A of the aldehyde oxidase-mediated metabolism of the experimental antitumour agent acridine carboxamide. Biochem Pharmacol. 1993 May 25;45(10):2159-62.

[4]. Inhibition of cytochrome P450 with proadifen alters the excitability of brain catecholamine-secreting neurons. Gen Physiol Biophys. 2022 May;41(3):255-262.

An inhibitor of drug metabolism and CYTOCHROME P-450 ENZYME SYSTEM activity.

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Metabolic stability of DMT in human liver microsomes: After incubation of DMT with HLM, DMT was rapidly metabolised with substantial amounts of IAA formed. No DMT-NO could be detected. When co-incubated with harmine (an inhibitor of both MAO-A and CYP2D6), DMT metabolism was completely inhibited. Co-incubation with the CYP2D6 specific inhibitor quinidine did not appear to affect DMT disappearance to any substantial degree. The general CYP inhibitor Proadifen/SKF-525A had a larger impact on DMT metabolism compared to quinidine (2.1-fold increase in half-life). Results are summarised in Table 1.[2]

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Summarizing, the inhibition of liver and/or brain CYP by Proadifen/SKF525 has an enhancing effect on the excitability of central noradrenalin, and inhibitory – on the excitability of central dopamine neurons. The stimulatory effect of the CYP inhibition on noradrenaline neuronal firing activity might be triggered, at least in part, by corticosterone-induced inhibition of 5-HT transmission. The effect of CYP inhibition on dopamine neuronal firing activity might be in torn secondary the putative activation of central noradrenaline transmission (Fig. 3). It is possible that the inhibitory effect of SKF525 on dopamine neurons, as well as the inhibitory effect of this CYP inhibitor on 5-HT neurons, observed on our previous study (Maj et al. 1981; Grinchii et al. 2018), at least partially explains the ability of SKF525 to diminish the efficacy of imipramine, a non-selective tricyclic antidepressant acting as 5-HT, noradrenaline, and dopamine reuptake inhibitor (Maj et al. 1981). Notably, Maj and colleagues reported the lack of diminishing effect of SKF525 on the efficacy of desipramine, an antidepressant drug primarily acting on noradrenaline system. Since glucocorticoids, as well as central catecholamines, are fundamental in stress, the interactions between circulating glucocorticoids and the excitability of catecholamine-secreting neurons might be of particular importance in pathophysiology of stress-related disorders. Further studies are however required to test this hypothesis. The main limitations of this study are the use of a nonselective CYP inhibitor and non-distinguishing between brain and hepatic CYP inhibition. The involvement of the inhibitory effect of Proadifen/SKF525 on the nitric oxide synthase (NOS; Sykes et al. 2016) cannot be excluded as well. In future studies, the effect of the selective inhibitors of the specific CYP subtypes, such as CYP3A1, CYP3A2, CYP3A4, and CYP3A5, which are fundamental in glucocorticoid metabolism (Peng et al. 2011), should be tested. [2]

C23H32CLNO2

389.9587

389.212

C, 70.84; H, 8.27; Cl, 9.09; N, 3.59; O, 8.21

62-68-0

302-33-0 (Parent)

65341

White to light yellow solid powder

460.8ºC at 760 mmHg

122-123ºC

132.3ºC

5.459

1

3

11

27

375

0

CCCC(C1=CC=CC=C1)(C2=CC=CC=C2)C(=O)OCCN(CC)CC.Cl

FHIKZROVIDCMJA-UHFFFAOYSA-N

InChI=1S/C23H31NO2.ClH/c1-4-17-23(20-13-9-7-10-14-20,21-15-11-8-12-16-21)22(25)26-19-18-24(5-2)6-3;/h7-16H,4-6,17-19H2,1-3H3;1H

2-(diethylamino)ethyl 2,2-diphenylpentanoate;hydrochloride

Proadifen hydrochloride; 62-68-0; Proadifen HCl; Propyladiphenin; SK&F 525-A; SKF 525A; RP 5171; Proadifen hydrochloride [USAN];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~128.22 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.33 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.33 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.33 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)