The study identifies ATTM as a slow-release sulfide donor. Its primary mechanism of action is the transient inhibition of mitochondrial cytochrome C oxidase (Complex IV), which modulates oxidative metabolism and reduces reactive oxygen species (ROS) production. It is also historically known as a copper chelator. [1]

The study references previous work showing that ATTM downregulates oxidative metabolism ex vivo in a dose-dependent manner. [1]
In an in vitro anoxia/reoxygenation study referenced, ATTM improved cell viability and reduced mitochondria-specific superoxide levels. [1]
The batch of ATTM used in this study was subjected to an in vitro sulfide-release test. Following standard incubation conditions (1 hour at physiological pH and temperature), it released between 3 and 4 parts per million (ppm) of H₂S, conforming to the predefined quality control criteria. [1]

Neuroprotective properties of intravenous (10 mg/kg) ammonium(VI) tetrathiomolybdate (ATTM) are noteworthy [1].

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In a rat model of transient middle cerebral artery occlusion, intravenous administration of ATTM at the time of reperfusion provided significant neuroprotection. [1]
ATTM treatment reduced brain infarct size by 68% at 24 hours and 54% at 7 days post-reperfusion compared to saline-treated controls. [1]
Functional outcomes were improved with ATTM. In the rotarod test, latency to first fall was significantly increased at both 24 hours and 7 days. In the open field test at 24 hours, ATTM-treated animals showed twice the number of line crossings and rearing events. [1]
ATTM attenuated oxidative damage in brain tissue, reducing protein carbonyls and thiobarbituric acid-reactive substances at both 24 hours and 7 days. [1]
ATTM enhanced antioxidant enzyme capacity, with significantly increased catalase activity at 24 hours and superoxide dismutase activity at 7 days. [1]
ATTM blunted the late pro-inflammatory response, with significant reductions in brain IL-1β, IL-6, and nitric oxide products at 7 days post-reperfusion. TNF-α levels were not significantly different at either time point. [1]
No drug-related deaths were observed; a similar number of animals from saline and ATTM groups succumbed to early demise due to overwhelming focal ischemia or anesthesia-induced airway obstruction. [1]

Sulfide Release Assay: The batch of ATTM was subjected to an in vitro sulfide-release test to ensure quality control. The material was incubated under standard conditions (1 hour at physiological pH and temperature), and the amount of H₂S released was measured in parts per million. The batch used in this study released between 3 and 4 ppm, which met the predefined criteria. [1]
Antioxidant Enzyme Activity Assays: Catalase activity was determined spectrophotometrically by monitoring the disappearance of hydrogen peroxide at 240 nm in a reaction medium containing H₂O₂, Triton X-100, and potassium phosphate buffer. One unit of catalase was defined as 1 μmol of H₂O₂ consumed per minute. Superoxide dismutase activity was measured by the inhibition of adrenaline autoxidation. A calibration curve using purified SOD was used to calculate specific activity, expressed as units per milligram of protein. [1]

Animal/Disease Models: Wistar rat[1]
Doses: 10 mg/kg
Route of Administration: intravenous (iv) (iv)injection
Experimental Results: Functional activities were improved 24 hrs (hrs (hours)) and 7 days after reperfusion, while the infarct size was Dramatically diminished.

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Stroke Model:** Transient middle cerebral artery occlusion was induced in anesthetized male Wistar rats for 90 minutes using an intraluminal filament technique. [1]
* **Treatment Regimen:** Immediately prior to reperfusion, animals were randomly assigned to receive an intravenous bolus of ATTM (10 mg/kg) or an equal volume of saline (2 ml/kg) administered over 1 minute. This was followed by a continuous intravenous infusion of ATTM (10 mg/kg/h) or saline (10 ml/kg/h) for 60 minutes. Anesthesia was maintained throughout ischemia and for 1 hour post-reperfusion. [1]
* **Outcome Assessments:** Separate cohorts of animals were used for histological (infarct size by TTC staining), functional (rotarod and open field tests), and molecular (brain tissue analysis for oxidative damage, antioxidant enzymes, cytokines, and nitric oxide) outcome measures at 24 hours and 7 days post-reperfusion. [1]
* **Euthanasia:** At the end of the experiments, animals were euthanized by terminal anesthesia with intraperitoneal sodium pentobarbitone. [1]

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Stroke Model: Transient middle cerebral artery occlusion was induced in anesthetized male Wistar rats for 90 minutes using an intraluminal filament technique. [1]
Treatment Regimen: Immediately prior to reperfusion, animals were randomly assigned to receive an intravenous bolus of ATTM (10 mg/kg) or an equal volume of saline (2 ml/kg) administered over 1 minute. This was followed by a continuous intravenous infusion of ATTM (10 mg/kg/h) or saline (10 ml/kg/h) for 60 minutes. Anesthesia was maintained throughout ischemia and for 1 hour post-reperfusion. [1]
Outcome Assessments: Separate cohorts of animals were used for histological (infarct size by TTC staining), functional (rotarod and open field tests), and molecular (brain tissue analysis for oxidative damage, antioxidant enzymes, cytokines, and nitric oxide) outcome measures at 24 hours and 7 days post-reperfusion. [1]
Euthanasia: At the end of the experiments, animals were euthanized by terminal anesthesia with intraperitoneal sodium pentobarbitone. [1]

The study does not report traditional ADME parameters. However, it provides important information related to the drug's disposition: [1]
Distribution: The paper references a study in sheep showing that following administration of ATTM, molybdenum (the transition metal core of ATTM) is selectively distributed and retained by many organs, including the brain. [1]
Cellular Uptake Mechanism: The study references previous work demonstrating that ATTM utilizes non-selective plasma membrane anion-exchanger-1 channels to gain access to intracellular compartments. This channel is also expressed at the blood-brain barrier, suggesting a potential route for CNS penetration. [1]
Metabolism: ATTM acts as a slow-release sulfide donor. Its sulfide release profile is dependent on biologically relevant factors; more acidic conditions and the presence of thiols (e.g., glutathione), both encountered intracellularly, help ensure that sulfide is preferentially released within this compartment. [1]

The study highlights the excellent safety profile of ATTM, noting it has been administered extensively to humans over many decades as a treatment for Wilson's disease. [1]
At the dose used in this study (10 mg/kg bolus + 10 mg/kg/h infusion), ATTM did not impact global hemodynamics or cardiac function (mean arterial blood pressure, cardiac output, and contractility) in previous studies. [1]
No drug-related deaths were observed during the experiment. Mortality was similar between the ATTM-treated and saline-treated groups and was attributed to the severity of the stroke model or anesthesia complications. [1]
The controlled, slow-release profile of sulfide from ATTM is presented as a safety advantage over basic sulfur salts, which release sulfide instantaneously. [1]

[1]. Neuroprotective effects of ammonium tetrathiomolybdate, a slow-release sulfide donor, in a rodent model of regional stroke. Intensive Care Med Exp. 2020 Apr 9;8(1):13.

Diammonium thiomolybdate is an ammonium salt with potential anti-angiogenic and antitumor activities. Studies have found that tetrathiomolybdate can deplete the body's copper reserves through an unknown mechanism. This drug has been shown to inhibit the activity of copper-containing enzymes, including superoxide dismutase 1 (SOD1) and cytochrome c oxidase (COX), which may contribute to its anti-angiogenic and antitumor effects.

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ATTM is a copper chelator historically used for the treatment of Wilson's disease. It is being repurposed as a slow-release sulfide donor for the treatment of ischemia-reperfusion injury. [1]
The drug consists of a transition metal (molybdenum) core with four covalently bound sulfur atoms. Scission of the metal-sulfur bonds enables it to act as a slow-release sulfide donor. [1]
The therapeutic hypothesis is that ATTM, by transiently inhibiting mitochondrial cytochrome C oxidase, modulates the surge in oxidative metabolism that occurs upon reperfusion. This reduces the damaging overproduction of ROS while still supporting cell viability and function. [1]
The study demonstrates that a single, acute dose of ATTM given at the time of reperfusion can improve both short-term (24h) and longer-term (7d) histological and functional outcomes in a preclinical stroke model. [1]
The favorable safety profile of ATTM in humans, combined with its efficacy in this and other preclinical reperfusion models, supports its potential development as an adjunct therapy for revascularization in stroke. [1]

H8MON2S4

260.278

261.862

15060-55-6

Tetrathiomolybdate;16330-92-0

15251598

Brown to black solid powder

>300 °C(lit.)

2.044

4

4

0

7

19.1

0

ZKKLPDLKUGTPME-UHFFFAOYSA-N

InChI=1S/Mo.2H3N.2H2S.2S/h;2*1H3;2*1H2;;

diazanium;bis(sulfanylidene)molybdenum;sulfanide

Tiomolibdate diammonium Ammonium tetrathiomolybdate Ammonium molybdenum sulfide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~5 mg/mL (~19.21 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 0.5 mg/mL (1.92 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 5.0 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: ≥ 0.5 mg/mL (1.92 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 5.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)