Pharmacokinetics of 1-nitrosomelatonin and detection by EPR using iron dithiocarbamate complex in mice

Abstract

The N-nitroso-derivative of melatonin, NOM (1-nitrosomelatonin), which has been demonstrated to be a NO* [oxidonitrogen*] donor in buffered solutions, is a new potential drug particularly in neurological diseases. The advantage of NOM, a very lipophilic drug, is its ability to release both melatonin and NO*, an easily diffusible free radical. In order to evaluate the distribution and the pharmacokinetics of NOM, [O-methyl-3H]NOM was administered to and followed in mice. A complementary method for monitoring NOM, EPR, was performed in vitro and ex vivo with (MGD)2-Fe2+ (iron-N-methyl-D-glucamine dithiocarbamate) complex as a spin trap. The behaviour of NOM was compared with that of GSNO (S-nitrosoglutathione), a hydrophilic NO* donor. In the first minutes following [O-methyl-3H]NOM intraperitoneal injection, the radioactivity was found in organs (6% in the liver, 1% in the kidney and 0.6% in the brain), but not in the blood. In both liver and brain, the radioactivity content decreased over time with similar kinetics reflecting the diffusion and metabolism of NOM and of its metabolites. Based on the characterization and the quantification of the EPR signal in vitro with NOM or GSNO using (MGD)2-Fe2+ complex in phosphate-buffered solutions, the detection of these nitroso compounds was realized ex vivo in mouse tissue extracts. (MGD)2-Fe2+-NO was observed in the brain of NOM-treated mice in the first 10 min following injection, revealing that NOM was able to cross the blood-brain barrier, while GSNO was not.

Figure 1. Pharmacokinetics of [³H]NOM in mice

[³H]NOM was injected intraperitoneally into mice, and the radioactivity was then measured in solubilized organs or tissues. The radioactivity recovered in the liver (□), kidney (●) and brain (▲) was measured at definite times for 1 h. The radioactivity is expressed as a percentage of the total injected radioactivity. Two independent experiments were carried out.

Figure 2. Relationship between the NOM concentration and the height of the EPR signal of (MGD)₂–Fe²⁺–NO complex

Peak-to-peak height of the first line of the (MGD)₂–Fe²⁺–NO signal in the mixture of (MGD)₂–Fe²⁺ and NOM plotted against time. At the initial time, the mixture of 50 mM MGD, 10 mM FeSO₄ and 10 μM NOM in Tris buffer, pH 7.5, was introduced in a flat cell at 298 K. A spectrum was recorded every 1 min using a Bruker EPR spectrometer. The inset shows a typical spectrum obtained after 60 min with 10 μM NOM. The same spectra were obtained immediately with 10 μM GSNO. The EPR triplet signal centred at g=2.04 with a nitrogen hyperfine coupling constant of 1.27 mT is characteristic of (MGD)₂–Fe²⁺–NO.

Figure 3. Effect of MGD on NOM decay

Spectrophotometric kinetics at 340 nm of 0.1 mM NOM in phosphate buffer, pH 7.5.

Figure 4. Typical spectra of livers isolated from NOM- and vehicle-treated mice

The upper spectra were obtained with livers isolated after i.p. injections of NOM (A) or vehicle (B). The liver was homogenized with a fresh (MGD)₂–Fe²⁺ complex, and was frozen 30 min later. The thin spectra (C and D) were recorded with mixtures of (MGD)₂–Fe²⁺ and NOM (1 or 10 μM), diluted twice in 0.1 M phosphate buffer, pH 7.5, as for tissue extracts. Samples were frozen in 5-mm quartz tubes in liquid nitrogen, and spectra were recorded on a Magnettech EPR spectrometer. Instrument settings are indicated in the Materials and methods section.

Figure 5. Typical EPR spectra of brains from NOM- and vehicle-treated mice

The upper spectra were obtained with brain isolated after i.p. injections of NOM (A) and vehicle (B) followed by perfusion. The brain was homogenized with a fresh (MGD)₂–Fe²⁺ complex, and was frozen 30 min later. Spectra were recorded at 77 K on a Magnettech EPR spectrometer. Instrument settings are indicated in the Materials and methods section.

Figure 6. Typical spectra obtained with brain and liver from GSNO- and vehicle-treated mice

The upper spectra were obtained with liver (A and B) and brain (C and D) isolated after i.p. injections of GSNO (A and C) or vehicle (B and D) followed by perfusion. The liver and the brain were homogenized with a fresh (MGD)₂–Fe²⁺ complex solution. Samples were frozen in 5-mm quartz tubes in liquid nitrogen, and spectra were recorded at 77 K on a Magnettech EPR spectrometer. Instrument settings are indicated in the Materials and methods section.

Figure 7. Intensities of the EPR signals obtained with livers isolated from NOM- or GSNO-treated mice and with NOM or GSNO phosphate buffer solutions

Columns correspond to the peak-to-peak heights of the two first lines of the EPR signal indicated in arbitrary units (means±S.D. out of at least three experiments). Mice were anaesthetized 7 min after i.p. injections of 0, 2.5 or 5 μmol of NOM, and 5 or 10 μmol of GSNO and then perfused. Livers were homogenized in a fresh (MGD)₂–Fe²⁺ solution. EPR measurements were made at 77 K. The inset shows in vitro experiments performed with mixtures of 10 mM (MGD)₂–Fe²⁺ complex and 1, 2, and 5 μM NOM or GSNO solution, diluted twice in phosphate buffer. Statistical significance (using Student's t test): **P<0.001; *P<0.05; NS, non-significant.

Figure 8. Intensities of the EPR signals obtained with brains isolated from NOM- or GSNO-treated mice

The series of columns correspond to ex vivo experiments. Mice were anaesthetized 7 min after i.p. injections of vehicle, 2.5 or 5 μmol of NOM, and 5 or 10 μmol of GSNO, and then perfused. Brains were homogenized in a fresh (MGD)₂–Fe²⁺ solution. EPR measurements were made at 77 K. When vehicle and GSNO were administered, the two first lines of the (MGD)₂–Fe²⁺–NO spectra were overlapped by a (MGD)₂–Cu²⁺ signal. When NOM was administered, the increase in the peak-to-peak height of the two first lines of the EPR spectra, expressed in arbitrary units (means±S.D. from at least three experiments), was attributed to (MGD)₂–Fe²⁺–NO. Statistical significance (using Student's t test): * P<0.05.