copper chelating agent; copper enzymes such as SOD1, cytochrome oxidase, and DβH

Copper(II) coordination by bis(cyclohexanone)oxalyldihydrazone (also known as Cuprizone), resulting in the formation of an intensely coloured blue complex, was first reported over 70 years ago. The cuprizone reaction has been employed in colourimetric tests for the presence of trace levels of copper. Cuprizone administration in C57BL/6 mice also leads to demyelination over time - a consequence that appears to be due to copper dyshomeostasis - and this has led to use of cuprizone as the leading method for toxicant-induced generation of an animal model of demyelination since its first use in the 1960s. Despite broad interest in cuprizone and its ability to bind copper there have been relatively few studies to structurally characterize the copper coordination properties of this ligand. In the absence of an aqueous medium, such as neat alcohol, copper and cuprizone exclusively form an amorphous green precipitate. Under aqueous conditions, where a large excess of cuprizone (relative to copper) is present, the blue complex that is synonymous with copper-cuprizone coordination is predominantly formed. The blue and green copper-cuprizone products demonstrate poor solubility and present challenges for conventional structure characterization methods, such as X-ray crystallography or nuclear magnetic resonance spectroscopy. By combining mass spectrometry, X-ray absorption spectroscopy, computational chemistry, and other techniques, a self-consistent picture of the copper coordination structures of the blue and green complexes is revealed - confirming that the blue complex is in the Cu(III) state, containing two hydrolyzed cuprizone ligands per metal centre, while the green complex represents an extended oligomeric complex, comprised of repeating Cu(II) centres that lie 4.8 Å apart and are bridged by unhydrolyzed cuprizone donors. [3]

The central nervous system of mice experiences a severe spongiform state when exposed to Cuprizone (0.5 mg/100 mg given to chow or freshwater feeding; epidermis), particularly in the brainstem and cerebellar white matter [1].

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Recent human studies suggest a role for altered oligodendrocytes in the pathophysiology of schizophrenia. Our recent animal study has reported some schizophrenia-like behaviors in mice exposed to Cuprizone (Xu et al., 2009), a copper chelator that has been shown to selectively damage the white matter. This study was to explore mechanisms underlying the behavioral changes in cuprizone-exposed mice and to examine effects of the antipsychotics haloperidol, clozapine and quetiapine on the changes in the mice. Mice given cuprizone for 14 days showed a deficit in the prepulse inhibition of acoustic startle response and higher dopamine in the prefrontal cortex (PFC), which changes were not seen in mice given cuprizone plus antipsychotics. Mice given cuprizone for 21 days showed lower spontaneous alternations in Y-maze, which was not seen in mice treated with cuprizone plus the antipsychotics. Mice given cuprizone for 28 days displayed less social interactions, which was not seen in mice given cuprizone plus clozapine/quetiapine, but was seen in mice given cuprizone plus haloperidol. Mice given cuprizone for 42 days showed myelin sheath loss and lower myelin basic protein in PFC, caudate putamen, and hippocampus. The white matter damage in PFC was attenuated in mice given cuprizone plus clozapine/haloperidol. But the white matter damage in caudate putamen and hippocampus was only attenuated by clozapine and quetiapine, not by haloperidol. These results help us to understand the behavioral changes and provide experimental evidence for the protective effects of antipsychotics on white matter damage in cuprizone-exposed mice.[4]

Male C57BL/6 mice (6-weeks old, 20 to 22 g) were used. After an acclimatization period of 10 days, the C57BL/6 mice were given 0.2% by weight Cuprizone/CPZ in the standard powdered rodent chow for 14–42 days, during which age-matched mice received the standard chow without CPZ and were used as controls. Three independent experiments were performed for HAL, CLZ, and QUE, respectively. Each experiment consisted of four animal groups designed to examine effects of CPZ on animals’ behaviors and white matter and effects of the antipsychotics in mice without or with CPZ-exposure. For example, HAL experiment included CNT, CPZ, HAL, and CPZ+HAL groups. CNT group mice ate normal rodent chow without CPZ and received no HAL; CPZ group mice ate CPZ-containing rodent chow and received no HAL; HAL group mice ate normal rodent chow without CPZ but received HAL treatment (1 mg/kg/day, i.p.); CPZ + HAL group mice ate CPZ-containing rodent chow and received HAL treatment. The experimental period was 42 days, during which mice were subjected to behavioral tests at indicated time points. For high-performance liquid chromatography (HPLC) analysis, two additional experiments were performed, in which the same treatments (CPZ ingestion and antipsychotics administration) continued for 12 and 21 days, respectively. Each of the experiments consisted of eight animal groups (CNT, CPZ, HAL, CLZ, QUE, CPZ+HAL, CPZ+CLZ, and CPZ+QUE; 5–8 mice/group). [4]

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Prepulse inhibition test[4]
PPI refers to the inhibition of a startle reflex produced by preceding the startling stimulus, or pulse, with a weak prepulse stimulus. This test provides an operational measure of sensory gating of subjects. In our recent study, CuprizoneCPZ-exposed mice showed PPI deficits on 14th and 21st days after CPZ-exposure (Xu et al., 2009). In this study PPI test was performed on 14th day after CPZ-exposure. As described in our recent study (Xu et al., 2009), each mouse was placed into a small Plexiglas cylinder within a large sound-attenuating chamber. The cylinder was seated upon a piezoelectric transducer, which allows vibrations to be quantified and displayed on a computer. The background sound levels (74–75 dB) and calibration of the acoustic stimuli were confirmed with a digital sound level meter. After a 5-min habituation period, PPI test sessions were conducted. Each session started and ended with five startle trials (40 ms; 120 dB), respectively. Between the starting and ending startle trials, there were eight identical blocks consisting of the following five trials: a no-stimulus trial, a startle trial, and three prepulse-startle trials, each of which had a pre-pulse stimulus (3, 6, or 12 dB above the background sound levels) prior to a startle stimulus (100 ms after the prepulse). The average inter-trial interval was 15 s. Measures were taken of the startle amplitude for each trial, defined as the peak response during a 65-ms sampling window starting from the onset of a startle stimulus. Levels of PPI at each prepulse sound level were calculated as 100 × (1-averaged response amplitude in trials with a prepulse stimulus and startle stimulus/averaged response amplitude in trials with the startle stimulus alone).

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Y-maze test [4]
The Y-maze is a simple two-trial recognition test for measuring spatial recognition memory. In a previous study (Oades et al., 1985), amphetamine reduced alternation dose-dependently and HAL pretreatment inhibited this effect, suggesting that this paradigm is useful for studying spatial working memory in animal models of schizophrenia. In our recent study, Cuprizone/CPZ-exposed mice showed lower spontaneous alternation in Y-maze on 14th, 21st, 28th, 35th, and 42nd days after CPZ-exposure (Xu et al., 2009). In this study Y-maze test was performed on 21st and 42nd days after CPZ-exposure. As described in our recent study (Xu et al., 2009), each mouse was placed at the end of one arm of a symmetrical Y-maze and allowed to move freely through the maze during an 8-min test period. The total number and series of arm entries were recorded. The number of overlapping entrance sequences (e.g., ABC, BCA) defines the number of spontaneous alternations.

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Social interaction test [4]
In our recent study, CuprizoneCPZ-exposed mice showed less social interaction on 28th, 35th, and 42nd days after CuprizoneCPZ-exposure (Xu et al., 2009). In this study social interaction test was performed on 28th day after CPZ-exposure. As described in our recent study (Xu et al., 2009), pairs of unfamiliar mice (that had been housed separately) of the same experimental group were placed in the center of an open-field box (56 cm × 56 cm × 31 cm) about 10 cm apart. A video camera was placed above the open-field box to monitor the animals’ movement. During a 10 min test period, the social interaction of the animals was defined when the tested mice were closer than 5 cm (between the center point of the back of a mouse and another one) for at least 0.2 s. Each mouse was used only one time. The results were recorded and analyzed automatically by the video tracking program SMART, which counted the amount of time spent near another mouse for two mice simultaneously. Although this method only measures closeness of subjects examined, it has been used in previous studies and the closeness is believed to reflect social interactions (Shi et al., 2003; Egashira et al., 2007), which is an animal correlate of social withdrawal seen in patients with schizophrenia (Weinberger, 1987; Abi-Dargham et al., 2000). Social behavioral deficit has been reported in various animal models of schizophrenia (Shi et al., 2003; Boucher et al., 2007; Lazar et al., 2008).

[1]. Suzuki K, et al. Status spongiosus of CNS and hepatic changes induced by cuprizone (biscyclohexanone oxalyldihydrazone). Am J Pathol. 1969 Feb;54(2):307-25.
[2]. Sanadgol N, et al. Alpha-lipoic acid mitigates toxic-induced demyelination in the corpus callosum by lessening of oxidative stress and stimulation of polydendrocytes proliferation. Metab Brain Dis. 2018 Feb;33(1):27-37.
[3]. M Jake Pushie, et al. Synthesis and structural characterization of copper-cuprizone complexes. Dalton Trans. 2022 Jun 29.
[4]. Haiyun Xu, et al. Behavioral and neurobiological changes in C57BL/6 mouse exposed to cuprizone: effects of antipsychotics. Front Behav Neurosci. 2010 Mar 18;4:8.

Cuprizon is an organooxygen compound and an organonitrogen compound. It is functionally related to an alpha-amino acid.
Copper chelator that inhibits monoamine oxidase and causes liver and brain damage.

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Multiple Sclerosis (MS), is a disease that degenerates myelin in central nervous system (CNS). Reactive oxygen species (ROSs) are toxic metabolites, and accumulating data indicate that ROSs-mediated apoptosis of oligodendrocytes (OLGs) plays a major role in the pathogenesis of MS under oxidative stress conditions. In this study, we investigated the role of endogenous antioxidant alpha-lipoic acid (ALA) as ROSs scavenger in the OLGs loss and myelin degeneration during Cuprizone (cup)-induced demyelination in the experimental model of MS. Our results have shown that ALA treatment significantly increased population of mature OLGs (MOG+ cells), as well as decreased oxidative stress (ROSs, COX-2 and PGE2) and apoptosis mediators (caspase-3 and Bax/Bcl2 ratio) in corpus callosum (CC). Surprisingly, ALA significantly stimulates population of NG2 chondroitin sulfate proteoglycan positive glia (NG2+ cells or polydendrocytes), from week 4 afterward. Accordingly ALA could prevents apoptosis, delays demyelination and recruits OLGs survival and regeneration mechanisms in CC. We conclude that ALA has protective effects against toxic demyelination via reduction of redox signaling, and alleviation of polydendrocytes vulnerability to excitotoxic challenge.[2]

C14H22N4O2

278.3501

278.174

C, 60.41; H, 7.97; N, 20.13; O, 11.50

370-81-0

9723

Typically exists as White to off-white solid at room temperature

1.3±0.1 g/cm3

210-214 °C(lit.)

1.626

0.85

2

4

2

20

373

0

O=C(C(N([H])/N=C1\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C\1([H])[H])=O)N([H])/N=C1\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C\1([H])[H]

DSRJIHMZAQEUJV-UHFFFAOYSA-N

InChI=1S/C14H22N4O2/c19-13(17-15-11-7-3-1-4-8-11)14(20)18-16-12-9-5-2-6-10-12/h1-10H2,(H,17,19)(H,18,20)

N,N'-bis(cyclohexylideneamino)oxamide

Cuprizone; 370-81-0; Bis(cyclohexanone)oxaldihydrazone; Ethanedioic acid, bis(cyclohexylidenehydrazide); Cuprizane; Oxalic acid bis(cyclohexylidenehydrazide); Cuprizon; Biscyclohexanone oxaldihydrazone;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~6.67 mg/mL (~23.96 mM)

Solubility in Formulation 1: ≥ 0.67 mg/mL (2.41 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 6.7 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 2: ≥ 0.67 mg/mL (2.41 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 6.7 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.)