Reaction mass of disodium [2,2'-(ethane-1,2-diylbis{[2-(hydroxy-kO)benzyl]imino-kN})diacetato-kO(4-)]zinc(2-) and sodium chloride

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well-documented publication, which meets basic scientific principles

Data source

Reference

Reference Type:

publication

Title:

Chelation of Mitochondrial Iron Prevents Seizure-Induced Mitochondrial Dysfunction and Neuronal Injury

Author:

Liang, L., Jarrett, S.G. and Patel M.

Year:

2008

Bibliographic source:

The Journal of Neuroscience, November 5, 2008 - 28(45):11550 –11556

Materials and methods

Objective of study:

distribution

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Adult male Sprague Dawley rats were injected subcutaneously (s.c.) with a single dose (12 mg/kg) of kainic acid (KA) dissolved in sterilized saline. HBED was injected at the dose of 75 µmol/kg, s.c. daily three times before KA and once after KA for a total of four injections. Thereafter the total brain iron levels were assayed.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis (2-hydroxybenzyl) ethylenediamine-N,N-diacetic acid

IUPAC Name:

N,N-bis (2-hydroxybenzyl) ethylenediamine-N,N-diacetic acid

Details on test material:

- Name of test material (as cited in study report): N,N-bis (2-hydroxybenzyl) ethylenediamine-N,N-diacetic acid (HBED)
- Molecular formula (if other than submission substance): C20H24N2O6
- Molecular weight (if other than submission substance): 424.89 g/mol
- Smiles notation (if other than submission substance): OC(=O)CN(CCN(CC(=O)O)Cc1ccccc1O)Cc1ccccc1O
- Substance type: chelate
- Physical state: solid

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 3 month of age
- Weight at study initiation: 300–350 g

Animal studies were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). All procedures were approved by the Institute Animal Care and Use Committee (IACUC) of the University of Colorado at Denver and Health Sciences Center (UCDHSC), which is fully accredited by the American Association for the Accreditation of Laboratory Animal Care.

Administration / exposure

Route of administration:

subcutaneous

Vehicle:

other: dissolved in dimethyl sulfoxide (DMSO) and diluted with sterilized phosphate buffered saline (PBS) to achieve the desired final concentration (1% DMSO)

Details on exposure:

Kainic acid (KA) and N,N-bis (2-hydroxybenzyl) ethylenediamine-N,N-diacetic acid (HBED) administration
Adult male Sprague Dawley rats (3 months of age, weighing 300–350 g) were injected subcutaneously (s.c.) with a single dose (12 mg/kg) of KA (Ocean Products International) dissolved in sterilized saline. HBED (Strem Chemicals) was injected at the dose of 75 µmol/kg, s.c. daily three times before KA and once after KA for a total of four injections. HBED was dissolved in dimethyl sulfoxide (DMSO) and diluted with sterilized phosphate buffered saline (PBS) to achieve the desired final concentration (1% DMSO).

Duration and frequency of treatment / exposure:

HBED (Strem Chemicals) was injected at the dose of 75 µmol/kg, s.c. daily three times before KA and once after KA for a total of four injections.

Doses / concentrations

Remarks:

Doses / Concentrations:
0 mol/kg, 75 µmol/kg

No. of animals per sex per dose / concentration:

3-6 animals

Control animals:

yes, concurrent vehicle

Details on dosing and sampling:

PHARMACOKINETIC STUDY (distribution)
- Tissues and body fluids sampled: brain tissue
- Time and frequency of sampling: rats were killed 7 d after KA administration

Results and discussion

Main ADME results

Type:

distribution

Results:

Intravascular compartment

Toxicokinetic / pharmacokinetic studies

Details on absorption:

No detailed information could be obtained in this study as the chemical was administered subcutaneously.

Details on distribution in tissues:

HBED penetrates the BBB and mitochondria - Before evaluating its neuroprotective effects, they determined the brain bioavailability of HBED after systemic administration. They measured HBED concentrations in the forebrain and mitochondrial fractions isolated from the hippocampi of rats at different time points after a single injection (75 µmol/kg, s.c). HBED levels were detected 1 h after administration, peaked at 3 h and remained stable for at least 6 h. Both tissue and mitochondrial levels remained 25% of initial values at the 24 h time-point after injection. These results suggest that HBED penetrates the BBB and is accessible to hippocampal mitochondria. The total iron levels in the rat hippocampus were measured with varying doses of HBED. Total iron levels measured by the ferrozine method in the hippocampus were decreased 30% after 4 or 5 daily injections with 75 µmol/kg. Increasing the injection frequency to 7 resulted in a 38% reduction of iron content compared with control animals but retarded weight gain. The dose of HBED of 75 µmol/kg was further optimized based on a dose–response study using chelatable iron changes in the mitochondria as an endpoint.

Transfer into organs

Transfer type:

blood/brain barrier

Observation:

distinct transfer

Metabolite characterisation studies

Metabolites identified:

no

Any other information on results incl. tables

HBED administration does not alter KA-induced behavioural seizure indices

To determine whether the HBED treatment protocol influenced KA-induced behavioural seizures, they evaluated seizure scores, latency to wet dog shakes and duration of SE. No significant differences were observed in behavioural seizure intensity, seizure latency time (44.0 +/-3.4 and 45.3 +/- 3.2 min for KA and KA-HBED, respectively; n = 12 rats per group) and seizure duration (384.2+/- 15.4 and 388.3+/- 16.1 min for KA and KA-HBED, respectively; n= 12 rats per group). The percentage of animals that received KA or KA-HBED treatment that did not go on to exhibit a stage 3 or 4 seizure and were thus eliminated from the study was 3.4% (2 of 59 rats) and 3.3% (2 of 60 rats), respectively. Mortality from KA alone and KA-HBED was 5.1% (3 of 59 rats) and 6.7% (4 of 60 rats), respectively. No significant differences in mortality rate were observed between KA and KAHBED groups.

HBED inhibits seizure-induced chelatable mitochondrial iron changes

Next, they assessed the effect of different doses of HBED on KA-induced increases in mitochondrial chelatable iron 24 h after KA injection in the four treatment groups. HBED doses of 75 and 150 µmol/kg, but not 37.5 µmol/kg, for a total 4 injections significantly decreased KA-induced changes in chelatable iron in the hippocampal mitochondrial fractions. Based on the bioavailability parameters and the ability to decrease total as well as chelatable iron, subsequent studies were conducted with the daily HBED dose of 75 µmol/kg for 4 d. The effects of HBED on SE-induced iron chelation were further verified using the RPA fluorescence method. RPA fluorescence quenching has been shown to be a selective indicator of intramitochondrial chelatable iron in both tissue and cells. HBED treatment significantly attenuated the KA-induced increase in mitochondrial chelatable iron detected by RPA fluorescence quenching. Analysis of RPA fluorescence density revealed that HBED treatment most prominently attenuated the increase in chelatable iron in the hippocampal CA3 region but also in the CA1 and dentate hilus.

HBED protects against SE-induced mitochondrial dysfunction

To determine whether mitochondrial iron chelation by HBED was sufficient to preserve mitochondrial targets after KA treatment, they analysed the integrity of mtDNA using a sensitive QPCR assay specific to the mitochondrial genome. KA-induced SE generated an approximate fivefold increase in mtDNA damage in the hippocampus compared with the control group at the 24 h time point. SE-induced mtDNA damage was significantly attenuated by HBED.

HBED inhibits SE-induced mitochondrial oxidative stress

To further determine the deleterious role of mitochondrial iron in SE-induced injury, they measured two additional indices of mitochondrial oxidative stress, the GSH, GSSG and 8-OHdG/2dG ratios. Twenty four hours after KA injection, hippocampal mitochondrial GSH levels were depleted ca. 40%; whereas the corresponding levels of GSSG were significant increased ca. 240% compared with controls. The changes in both GSH and GSSG were significantly restored by HBED. The level of DNA oxidation measured by the ratios of 8-OHdG/2dG, was enhanced > 3-fold by KA-induced SE and attenuated ca. 50% by HBED in the hippocampal mitochondrial fractions.

HBED protects against SE-induced neuronal degeneration

Finally, they evaluated the neuroprotective effects of HBED by Fluoro-Jade B fluorescence, a sensitive marker assessing degeneration of neuronal cell bodies, dendrites, axons, or terminals. Previous reports in the literature have demonstrated that Fluoro-Jade B is a more sensitive, reliable and definitive marker of neuronal degeneration than silver staining techniques. No significant Fluoro-Jade B staining indicative of degeneration was observed in any brain region of control animals. However, significant staining (degeneration) was observed in the cell bodies and terminals in the hippocampal CA1, CA3 and hilar regions but not granule cell layer of rats injected with KA beginning ca. 48 h and peaking at ca. 7 d after injection. The percentage of relative fluorescence density quantified by Image J was increased ca. 190, ca. 240 and ca. 265% in the CA1, CA3 and hilus, respectively in the KA vs control groups. Significant protection of neuronal degeneration was observed in the KAHBED group compared with KA alone indicating a neuroprotective effect of HBED.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: Intravascular distribution without side effects
Measurement of brain HBED levels after systemic administration confirmed its penetration in hippocampal mitochondria.

Executive summary:

Liang and colleagues investigated the distribution of HBED in rats (Liang, 2008). Chelatable iron is an important catalyst for the initiation and propagation of free radical reactions and implicated in the pathogenesis of diverse neuronal disorders. Studies have shown that mitochondria are the principal source of reactive oxygen species production after status epilepticus (SE). It was investigated whether SE modulates mitochondrial iron levels and whether consequent mitochondrial dysfunction and neuronal injury could be ameliorated with a cell-permeable iron chelator. Kainate (KA) induced SE resulted in a time-dependent increase in chelatable iron in mitochondrial but not cytosolic fractions of the rat hippocampus. Systemically administered N,N-bis (2-hydroxybenzyl) ethylenediamine-N,N-diacetic acid (HBED), a synthetic, BBB permeable iron chelator, ameliorated SE-induced changes in chelatable iron, mitochondrial oxidative stress (8-hydroxy-2 deoxyguanosine and glutathione depletion), mitochondrial DNA integrity and hippocampal cell loss. HBED significantly attenuates SE induced hippocampal neuronal damage. Measurement of brain HBED levels (brain bioavailability of HBED after systemic administration, measured in forebrain tissue and hippocampal mitochondria) after systemic administration of 75 µmol/kg, s.c. confirmed its penetration in hippocampal mitochondria. HBED levels were detected 1 h after administration, peaked at 3 h and remained stable for at least 6 h. Moreover, both tissues and mitochondrial HBED levels remained 25 % of the peak values until 24 h after injection. These results suggest that HBED penetrates the BBB and is accessible to hippocampal mitochondria. Total iron levels in the hippocampus were decreased 30% after 4 or 5 daily injections with 75 µmol/kg HBED. Increasing the injection frequency to 7 resulted in a 38% reduction of iron content compared with control animals but retarded weight gain. These results suggest a role for mitochondrial iron in the pathogenesis of SE-induced brain damage and subcellular iron chelation as a novel therapeutic approach (neuroprotective effects of HBED by inhibition of SE induced mitochondrial oxidative stress) for its management. However, HBED’s inability to influence behavioural seizure parameters produced by KA suggests that its mechanism of neuroprotection is not related to interference with acute seizure initiation and a direct antioxidant action of HBED cannot be ruled out.