Ciproxifan is a well-investigated histamine H3 receptor (H3R) inverse agonist/antagonist, showing an exclusively high species-specific affinity at rodent compared to human H3R. It is well studied as reference compound for H3R in rodent models for neurological diseases connected with neurotransmitter dysregulation, e.g. attention deficit hyperactivity disorder or Alzheimer’s disease. In a screening for potential monoamine oxidase A and B inhibition ciproxifan showed efficacy on both enzyme isoforms. Further characterization of ciproxifan revealed IC50 values in a micromolar concentration range for human and rat monoamine oxidases with slight preference for monoamine oxidase B in both species. The inhibition by ciproxifan was reversible for both human isoforms. Regarding inhibitory potency of ciproxifan on rat brain MAO, these findings should be considered, when using high doses in rat models for neurological diseases. As the H3R and monoamine oxidases are all capable of affecting neurotransmitter modulation in brain, we consider dual targeting ligands as interesting approach for treatment of neurological disorders. Since ciproxifan shows only moderate activity at human targets, further investigations in animals are not of primary interest. On the other hand, it may serve as starting point for the development of dual targeting ligands.
Ciproxifan (cyclopropyl 4-(3-(1H-imidazol-4-yl)propyloxy)phenyl methanone) is a well characterized species-specific histamine H3 receptor (H3R) inverse agonist/antagonist (Fig. 1). It shows exclusively high affinity at rodent H3R in a sub-nanomolar range (Ki (rH3R) = 0.4–6.2 nM and Ki (mH3R) = 0.5–0.8 nM), while binding to human H3R is only moderate (Ki = 46–180 nM) with negligible selectivity e.g. over human adrenergic α2A and α2C receptors1,2,3 (Table 1). Ciproxifan’s inverse agonism/antagonism at histamine H3 receptors is manifested in improvement of wakefulness and attention in vivo4,5. It is commonly used as reference H3R antagonist, e.g. in rodent models studying cognitive impairment6, Alzheimer’s disease7 or attention deficit hyperactivity disorder (ADHD)8. It was also tested in animal models for schizophrenia9, sleeping disorders10 or most recently autism11.
The H3R (for review, e.g. see Sander et al.5 or Gemkow et al.12), a G-protein coupled receptor displaying constitutive activity (basal activity without binding of an agonist), inhibits the release of several neurotransmitters like dopamine, histamine, serotonin or acetylcholine4. In consequence, inverse agonism/antagonism of the H3 receptor leads to accelerated release of mentioned neurotransmitters which is why H3 receptor inverse agonists/antagonists like ciproxifan are recognized as promising therapeutics for treatment of several neuropathological diseases13. During a screening for monoamine oxidase A (MAO A) and B (MAO B) inhibitors, ciproxifan was found to be an inhibitor for both enzyme isoforms. MAOs are expressed in neurons and glial cells, localized in the cell on the outer membrane of mitochondria and critically involved in degradation of neurotransmitters in the brain. In humans MAO A is predominantly found in adrenergic, catecholaminergic and dopaminergic neurons and deactivates serotonin, dopamine, norepinephrine and epinephrine. Human MAO B participates in dopamine degradation and is mainly expressed in serotonergic neurons and glial cells14,15. Therefore, MAO inhibitors are frequently investigated for treatment of depression and Parkinson’s disease16. In this study, we further investigated ciproxifan’s capability to inhibit human MAO A and MAO B in vitro by determination of IC50 values and reversibility of its inhibition.
IC50 determinations for human MAO
The IC50 values of ciproxifan for human membrane-bound MAO (hMAO) were measured spectrophotometrically using kynuramine (KYN) and benzylamine (BZA) as MAO B substrates, while for MAO A only KYN was used. We found IC50 values for ciproxifan in a micromolar range (IC50, MAO A = 11 μM and IC50, MAO B = 2 μM), showing an about 5-fold higher preference for MAO B (IC50, MAO B/IC50, MAO A = 0.2) (Fig. 1, Table 2).
l -Deprenyl, clorgyline, safinamide and moclobemide were tested as reference compounds using the same spectrophotometric method. The irreversible MAO B selective inhibitor l -deprenyl showed an IC50 value of 37 nM for MAO B (Table 2). For clorgyline, an irreversible MAO A selective inhibitor, an IC50 value of 8 nM for MAO A were found. The reversible inhibitors safinamide and moclobemide gave IC50 values of 49 nM (MAO B) and 568 μM (MAO A), respectively (Table 2).
IC50 determinations for rat brain MAO
The IC50 values of ciproxifan for rat brain MAO (rMAO) were obtained radiometrically using serotonin (5-HT) and phenylethylamine (PEA) as MAO A and MAO B substrates, respectively. Similar to hMAO, ciproxifan displayed IC50 values in the micromolar concentration range (IC50, MAO A = 38 μM and IC50, MAO B = 15 μM), again with slight preference for MAO B (IC50, MAO B/IC50, MAO A = 0.4) (Table 2).
Reversibility of human MAO inhibition
In order to determine whether ciproxifan shows a reversible or irreversible inhibition type, dilution experiments using the spectrophotometric assay were performed, where hMAOs were preincubated with ciproxifan (10 × IC50). After preincubation probes were diluted 100-fold, measured at saturated substrate conditions and the remaining enzyme activity was compared to that of MAO preincubated without ciproxifan. For both hMAO isoforms no considerable decrease in enzyme activity after preincubation with ciproxifan compared to control (set to 100%) were observed, suggesting a reversible inhibition type (Table 2). The remained enzyme activities for hMAO A and hMAO B preincubated with ciproxifan were 107.7 ± 3.4% and 91.4 ± 9.7%, respectively. In order to verify the test procedure, l -deprenyl was tested in the same manner showing decreased remaining enzyme activity of hMAO B (51.1 ± 2.9%) after preincubation.
Ciproxifan is frequently used as the reference histamine H3 receptor (H3R) antagonist in rodent models for neurological diseases like cognition6, Alzheimer’s disease7 or sleep-wake disorders10,17, because of its explicit high affinity and efficacy in rodent H3R (Ki < 1.0 nM) which is about 30- to 100-fold lower than that at the human H3R. In our study we showed an additional property of ciproxifan. It inhibits human and rat MAO A and MAO B reversibly in a micromolar concentration range with a slight preference for MAO B.
We consider a combined activity pattern of ligands at H3R and MAO as new interesting approach for the treatment of neurological diseases. These are often associated with a neurotransmitter dysregulation, assumed to be adjustable by H3R blockade12. Neurotransmitter levels could be also modulated by inhibition of their degradation. MAOs are enzymes involved in oxidative deamination of neurotransmitters in neuronal cells after reuptake from the synaptic cleft. Therefore, deactivation of MAO A or MAO B is a principle well established in therapy of neurological disorders like depression and Parkinson’s disease18. Additionally, MAOs are thought to promote oxidative stress when highly expressed in neuronal tissues. This can force increased neuronal cell death, a condition observed in Alzheimer’s disease19. Thus, we hypothesize that reversible or more probably irreversible MAO inhibitors and H3R inverse agonists/antagonists can have overlapping pharmacological utilities, suggesting ligands, interacting with both targets, as promising candidates for treatment of neurodegenerative diseases. Concerning ciproxifan, which displays IC50 values for both hMAO isoforms only in a micromolar range, we found its inhibition far too low for therapeutic efficacy in humans, which is anyway limited by its low affinity at human H3R. For example, safinamide, a MAO B selective reversible inhibitor most recently approved as first add-on treatment of Parkinson’s disease, is active in submicromolar concentration ranges (IC50 = 0.048–0.112 μM for hMAO B; IC50, MAO B/IC50, MAO A < 0.00120,21,22). Additionally, imidazole-containing drugs like ciproxifan are potential inhibitors of cytochrome P450 enzymes by coordination of the heme iron atom23. So, it may only serve as prospective starting point for investigation of dual targeting ligands in the future. Since ciproxifan is frequently used in rodent models for several neurological diseases, its MAO inhibition should be taken into consideration in retrospect or in the future using rodent models as possible accompanying effect. Ciproxifan has about four orders of magnitude higher activity at rH3R compared to the in-cell target rMAO. However, with a calculated log P value of 2.7624 we assume good membrane penetration by ciproxifan23. Additionally, it could be shown that ciproxifan can reach brain concentrations up to approximately 10 μM when applied i.p. to rats17, evidencing that partial rMAO inhibition is at least conceivable under test conditions.
Taken together, the moderate and reversible MAO inhibitory properties of ciproxifan are interesting newly described properties which may interact with some previous animal screening data at high ciproxifan dosages. It can still be taken as a reference H3R antagonist, but its MAO A and MAO B inhibitory properties may be considered in retrospect on high dosage screenings.
Nevertheless, ciproxifan may serve as starting point for the design of dual targeting ligands combining H3 receptor inverse agonism/antagonism and MAO inhibition, possibly favourable in treatment various neurological diseases. Since ciproxifan inhibited rat brain MAO also in micromolar concentration ranges, its activity has to be take into consideration in the future as a possible accompanying effect, when using it in rat models for neurological diseases. In retrospect, some of its effects explored in different species were probably contributed by its MAO A/B inhibitory properties.
All authors confirmed that all methods were carried out in accordance with relevant guidelines and regulations.
Spectrophotometric IC50 determination using human MAO
Enzyme studies were carried out using human recombinant membrane-bound MAO A and MAO B (Sigma-Aldrich, St. Louis, MO). Pipetting of assays were fully automated using a pipetting robot in a total assay volume of 100 μL or performed manually in a total assay volume of 200 μL. IC50 values were obtained by measuring enzymatic conversion rates at inhibitor concentrations between 10−9 M and 10−3 M in the presence of kynuramine (KM = 40 μM for MAO A and KM = 25 μM for MAO B) or benzylamine (KM = 165 μM) using 2 × KM substrate concentrations, while reactions were started by addition of MAO A (10 ng μL−1) or MAO B (12.5 ng μL−1). For optimal enzyme activity all assays were carried out under potassium phosphate buffered conditions (50 mM, pH = 7.4). Initial velocities were determined spectrophotometrically by a microplate reader at 30 °C by following product formation of 5-hydroxyquinoline and benzaldehyde at 316 nm and 250 nm, respectively, over a period of at least 30 minutes (interval of 20–30 seconds). Initial velocities, expressed as mAU min−1, were obtained from the linear phase of product formation (see Supplementary Information, Fig. 1). Data were analysed using GraphPad PRISM version 6. For IC50 determinations initial velocities for each experiment were normalized (expressed as percentage), plotted against inhibitor concentrations and fitted using the non-linear regression “log inhibitor vs. response (three parameters)”. IC50 values were determined in at least three independent experiments, each performed at least in duplicates.
Radiometric IC50 determination using rat brain MAO
Wistar male rats were sacrificed, the brains were quickly excised from the skulls, cleaned of residual meninges and frozen on dry ice. For each experiment the crude homogenates prepared from pooled brain from three rats were used. Enzyme activity of MAO A or MAO B was measured using radioactive substrate (PerkinElmer/NEN): serotonin (5-[2-14C]-hydroxytryptamine binoxalate) or β-[ethyl-1-14C]-phenyl-ethyl-amine hydrochloride (PEA), respectively, with the procedure described by Fowler and Tipton 198125 with some modification according to Gómez et al.26. IC50 values were determined using six different concentrations of ciproxifan between 10−9 M and 5 × 10−4 M at fixed concentration of substrate (200 μM serotonin or 20 μM PEA). For IC50 determinations values were normalized (expressed as percentage), plotted against inhibitor concentrations and fitted using the non-linear regression “log inhibitor vs. response (three parameters)”. IC50 values were determined in three independent experiments, each performed in duplicates.
Reversibility of human MAO inhibition
Reversibility of hMAO inhibition by ciproxifan was assessed by dilution experiments. Supplied MAOs (5 mg mL−1 in potassium phosphate 100 mM, sucrose 0.25 M, EDTA 0.1 mM, glycerol 5%) were preincubated with inhibitor (10 × IC50) or water at 30 °C for 15 minutes in a water bath. The inhibitor volume represented one-tenth of the total preincubation volume. After preincubation, probes were 100-fold diluted with potassium phosphate buffer (100 mM, pH = 7.4) to yield a final concentration of 12.5 ng μL−1 for the enzyme. Enzymatic conversion rates were determined in the presence of kynuramine for hMAO A and benzylamine for hMAO B at saturating substrate concentrations (10 × KM) under potassium phosphate buffer conditions (50 mM, pH = 7.4). Spectrophotometric measurements were carried out as described for IC50 determinations over a period of at least 30 minutes for hMAO A and hMAO B, respectively (interval of 20–30 seconds). Initial velocities, obtain from the linear phase of product formation, of hMAO preincubated with inhibitor (10 × IC50) were compared to hMAO preincubated with water (control) to define reversible or irreversible inhibition mode (see Supplementary Information, Fig. 2). Initial velocities, expressed as mAU min−1, were normalized and given as percent of control.
How to cite this article: Hagenow, S. et al. Ciproxifan, a histamine H3 receptor antagonist, reversibly inhibits monoamine oxidase A and B. Sci. Rep. 7, 40541; doi: 10.1038/srep40541 (2017).
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Ligneau, X. et al. Distinct pharmacology of rat and human histamine H(3) receptors: role of two amino acids in the third transmembrane domain. Br J Pharmacol 131, 1247–1250 (2000).
Ligneau, X. et al. Neurochemical and behavioral effects of ciproxifan, a potent histamine H3-receptor antagonist. J. Pharmacol. Exp. Ther. 287, 658–666 (1998).
Esbenshade, T. & Krueger, K. Two novel and selective nonimidazole histamine H3 receptor antagonists A-304121 and A-317920: I. In vitro pharmacological effects. J. Pharmacol. Exp. Ther. 305, 887–896 (2003).
Leurs, R., Bakker, R. A., Timmerman, H. & de Esch, I. J. P. The histamine H3 receptor: from gene cloning to H3 receptor drugs. Nat. Rev. Drug Discov. 4, 107–120 (2005).
Sander, K., Kottke, T. & Stark, H. Histamine H3 receptor antagonists go to clinics. Biol. Pharm. Bull. 31, 2163–2181 (2008).
Fox, G. B. et al. Cognition enhancing effects of novel H3 receptor (H3R) antagonists in several animal models. Inflamm Res 53, S49–S50 (2004).
Bardgett, M. E., Davis, N. N., Schultheis, P. J. & Griffith, M. S. Ciproxifan, an H3 receptor antagonist, alleviates hyperactivity and cognitive deficits in the APPTg2576 mouse model of Alzheimer’s disease. Neurobiol. Learn. Mem. 95, 64–72 (2011).
Fox, G. B. et al. Effects of histamine H3 receptor ligands GT-2331 and ciproxifan in a repeated acquisition avoidance response in the spontaneously hypertensive rat pup. Behav. Brain Res. 131, 151–161 (2002).
Mahmood, D., Khanam, R., Pillai, K. K. & Akhtar, M. Protective effects of histamine H3 -receptor ligands in schizophrenic behaviors in experimental models. Pharmacol. Reports 64, 191–204 (2012).
Gondard, E. et al. Enhanced histaminergic neurotransmission and sleep-wake alterations, a study in histamine H3-receptor knock-out mice. Neuropsychopharmacology 38, 1015–1031 (2013).
Baronio, D. et al. Effects of an H3R antagonist on the animal model of autism induced by prenatal exposure to valproic acid. PLoS One 10, 1–11 (2015).
Gemkow, M. J. et al. The histamine H3 receptor as a therapeutic drug target for CNS disorders. Drug Discov. Today 14, 509–515 (2009).
Passani, M. B. & Blandina, P. Histamine receptors in the CNS as targets for therapeutic intervention. Trends Pharmacol. Sci. 32, 242–249 (2011).
Wang, C. C., Billett, E., Borchert, A., Kuhn, H. & Ufer, C. Monoamine oxidases in development. Cell. Mol. Life Sci. 70, 599–630 (2013).
Ramsay, R. R. Monoamine oxidases: the biochemistry of the proteins as targets in medicinal chemistry and drug discovery. Curr. Top. Med. Chem. 12, 2189–2209 (2012).
Youdim, M. B. H. & Riederer & P. F. In Handbook of Clinical Neurology (eds. Koller, W. C. & Melamed, E. ) 84, 93–120 (Elsevier B.V., 2007).
Le, S., Gruner, J. a, Mathiasen, J. R., Marino, M. J. & Schaffhauser, H. Correlation between ex vivo receptor occupancy and wake-promoting activity of selective H3 receptor antagonists. J. Pharmacol. Exp. Ther. 325, 902–909 (2008).
Finberg, J. P. M. Update on the pharmacology of selective inhibitors of MAO-A and MAO-B: focus on modulation of CNS monoamine neurotransmitter release. Pharmacol. Ther. 143, 133–152 (2014).
Xie, S. et al. Synthesis and evaluation of selegiline derivatives as monoamine oxidase inhibitor, antioxidant and metal chelator against Alzheimer’s disease. Bioorg. Med. Chem. 23, 3722–3729 (2015).
Mostert, S., Petzer, A. & Petzer, J. P. Inhibition of monoamine oxidase by benzoxathiolone analogues. Bioorganic Med. Chem. 26, 1200–1204 (2016).
Choi, J. W. et al. Synthesis of a series of unsaturated ketone derivatives as selective and reversible monoamine oxidase inhibitors. Bioorg. Med. Chem. 23, 6486–6496 (2015).
Stocchi, F. & Torti, M. Adjuvant therapies for Parkinson’s disease: critical evaluation of safinamide. Drug Des. Devel. Ther. 10, 609–618 (2016).
Rydzewski, R. M. A Chemist’s Guide to Biotech and Pharmaceutical Research. (Elsevier, 2008).
Molsoft L. L. C., Drug-Likeness and Molecular Property Prediction. http://molsoft.com/mprop/ (accessed Nov. 3, 2016).
Fowler, C. J. & Tipton, K. F. Concentration dependence of the oxidation of tyramine by the two forms of rat liver mitochondrial monoamine oxidase. Biochem. Pharmacol. 30, 3329–3332 (1981).
Gómez, N., Balsa, D. & Unzeta, M. A comparative study of some kinetic and molecular properties of microsomal and mitochondrial monoamine oxidase. Biochem. Pharmacol. 37, 3407–3413 (1988).
Yao, B. B., Sharma, R., Cassar, S., Esbenshade, T. A. & Hancock, A. A. Cloning and pharmacological characterization of the monkey histamine H3 receptor. Eur. J. Pharmacol. 482, 49–60 (2003).
Medhurst, A. D. et al. GSK189254, a novel H3 receptor antagonist that binds to histamine H3 receptors in Alzheimer’s disease brain and improves cognitive performance in preclinical. J. Pharmacol. Exp. Ther. 321, 1032–1045 (2007).
Wulff, B. S., Hastrup, S. & Rimvall, K. Characteristics of recombinantly expressed rat and human histamine H3 receptors. Eur. J. Pharmacol. 453, 33–41 (2002).
Rouleau, A. et al. Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms. J. Neurochem. 90, 1331–1338 (2004).
Zhao, C. et al. The alkaloid conessine and analogues as potent histamine H3 receptor antagonists. J. Med. Chem. 51, 5423–5430 (2008).
Alcaro, S. et al. Chromone-2- and -3-carboxylic acids inhibit differently monoamine oxidases A and B. Bioorganic Med. Chem. Lett. 20, 2709–2712 (2010).
Chaurasiya, N. D., Ibrahim, M. A., Muhammad, I., Walker, L. A. & Tekwani, B. L. Monoamine oxidase inhibitory constituents of propolis: Kinetics and mechanism of inhibition of recombinant human MAO-A and MAO-B. Molecules 19, 18936–18952 (2014).
Support was kindly provided by the EU COST Actions CM1103 and CA15135 as well by DFG INST 208/664–1 FUGG and the Polish National Science Centre HARMONY 2012/04/M/N24/00212.
The authors declare no competing financial interests.
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Hagenow, S., Stasiak, A., Ramsay, R. et al. Ciproxifan, a histamine H3 receptor antagonist, reversibly inhibits monoamine oxidase A and B. Sci Rep 7, 40541 (2017). https://doi.org/10.1038/srep40541
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