Intranasal delivery of dodecyl creatine ester alleviates motor deficits and increases dopamine levels in a 6-OHDA rat model of parkinsonism

Abstract

Introduction: Creatine has been recognized not only as an energy buffer but also for its antioxidant, antiapoptotic, and anti-excitotoxic properties, making it of interest as a neuroprotective agent. Oral creatine monohydrate supplementation is ineffective due to poor brain and neuronal distribution and optimized forms of creatine deserve to be studied. Thus, dodecyl creatine ester (DCE), named CBT101, is a prodrug of creatine created for this purpose. When administered nasally it can follow the nose-to-brain pathway to deliver creatine to neuronal cells after passive diffusion across membranes. In this study, the therapeutic efficacy of intranasal DCE treatment was demonstrated in a 6-OHDA-intoxicated rat model, which is relevant to neurodegenerative diseases such as Parkinson's disease.

Methods: 6-OHDA-intoxicated rats received DCE (13.3 mg/kg/day) or a vehicle intranasally for 5 weeks and were compared to a sham group. Imbalance in dopamine between the two hemispheres was assessed using the amphetamine-induced turning test after 3 weeks and sensorimotor performance using the beam walking test after 4 weeks, with ongoing treatment.

Results and discussion: Five weeks after 6-OHDA intoxication, daily intranasal DCE treatment improved sensorimotor performance, striatal dopamine concentration, and modulated striatal pro-BDNF/BDNF balance and neurofilament expression both in plasma and in the striatum. These observations highlight DCE's potential as a therapeutic strategy for neurodegenerative diseases characterized by energy deficiency and major mitochondrial dysfunction.

Keywords: 6-hydroxydopamine; Parkinson’s disease; dodecyl creatine ester; intranasal drug delivery; mitochondrial dysfunction; motor behavior.

Figure 1

Experimental design. The Parkinson’s disease (PD) mouse model was induced by stereotactic 6-OHDA administration into the right medial forebrain bundle. The intranasal DCE-δ3 treatment was delivered to the rat starting on 1 day before 6-OHDA intoxication and daily for 5 weeks at a dose of 13.3 mg/kg/day. The sensorimotor tests were carried out in the 4th and 5th weeks. After that, the rats were sacrificed and biochemical and molecular analyses were carried out.

Figure 2

Amphetamine-induced rotational behaviors and beam walking tests in 6-OHDA-intoxicated rats after intranasal treatment with DCE-δ3. (A) Body weight monitoring in 6-OHDA-intoxicated rats after stereotactic administration up to the end of IN DCE-δ3 treatment for 5 weeks. Weight is expressed as a percentage from the day of 6-OHDA injection (D0) and is shown as the mean in each group ± SEM, n = 10 to 12 rats (B) Amphetamine-and apomorphine-induced turning test in 6-OHDA rats after 4 weeks of intranasal treatment with DCE-δ3. Results are expressed as the number of rotations per 15 min after challenge with amphetamine or apomorphine. Tests were performed after 3 weeks of treatment (C) Beam walking test in 6-OHDA rats after 4 weeks of intranasal treatment with DCE-δ3. Performance evaluated by number of segments crossed, crossing time in seconds, and walking score. Tests were performed after 4 weeks of treatment. Each data point represents one animal, with a median of n = 10 to 12 rats. Statistical comparison between sham rats, 6-OHDA-intoxicated rats treated with the vehicle, and 6-OHDA-intoxicated rats treated with CBT101-δ3 for a month was performed by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05; ** p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3

Concentration of dopamine, nicotinamide adenine dinucleotide and serotonin in right striatum of 6-OHDA-intoxicated rats after intranasal treatment with DCE-δ3. Dopamine (A), nicotinamide adenine dinucleotide (NAD) (B) and serotonin (5HT) (C) measured by HPLC in 6-OHDA-intoxicated rat striatum with or without IN DCE-δ3 treatment for 5 weeks. Results presented as concentrations in the 6-OHDA-intoxicated striatum (right hemisphere) and ratio of concentration in the right striatum injected with 6-OHDA over the left. Statistical comparison was performed by the Mann–Whitney test. **p < 0.01. n = 5–6 animals.

Figure 4

Modulation of neurofilament expression in the striatum and plasma of 6-OHDA-intoxicated rats after 5 weeks of intranasal treatment with DCE-δ3. Western blot analysis of proteins levels Nf-L (A) and Nf-M and Nf-H (C) in the striatum of the sham, 6-OHDA/vehicle and 6-OHDA/DCE-δ3 groups, 5 weeks after the 6-OHDA intoxication and the intranasal treatment. Graphs showing densitometric analysis of intensity of immunoblots. Values normalized to the sham group set at 100%. Circulating Nf-L were measured in plasma (B) by the Simoa^R Technology (from Quanterix). Results expressed in pg/mL of plasma. Statistical comparison was performed by the Kruskal-Wallis test followed by Dunn’s uncorrected post hoc test. * p < 0.05; **p < 0.01. n = 5–6 animals for western blotting and n = 10–12 for circulating NF-l quantification in plasma.

Figure 5

Modulation of pro-BDNF/BDNF balance in the striatum of 6-OHDA-intoxicated rats after 5 weeks of intranasal treatment with DCE-δ3. (A) Representative western immunoblot of pro-BDNF and BDNF in the striatum of the sham, 6-OHDA/vehicle and 6-OHDA/DCE-δ3 groups, 5 weeks after the 6-OHDA intoxication and the intranasal treatment. (B) Graphs showing densitometric analysis of the intensity of immunoblots. Values were normalized to the sham group set at 100%. Statistical comparison was performed by the Kruskal-Wallis test followed by Dunn’s uncorrected post hoc test. * p < 0.05; **p < 0.01. n = 5–6 animals.