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. 2019 Jun;14(12):1579-1593.
doi: 10.2217/nnm-2019-0059. Epub 2019 Apr 30.

Dodecyl creatine ester-loaded nanoemulsion as a promising therapy for creatine transporter deficiency

Gabriela Ullio-Gamboa  1 , Kenea C Udobi  2 , Sophie Dezard  3 , Marla K Perna  2 , Keila N Miles  2 , Narciso Costa  1 , Frédéric Taran  3 , Alain Pruvost  4 , Jean-Pierre Benoit  5 , Matthew R Skelton  2 , Pascale de Lonlay  6 , Aloïse Mabondzo  1
Affiliations

Dodecyl creatine ester-loaded nanoemulsion as a promising therapy for creatine transporter deficiency

Gabriela Ullio-Gamboa et al. Nanomedicine (Lond). 2019 Jun.

Abstract

Creatine transporter (CrT) deficiency is an X-linked intellectual disability caused by mutations of CrT. Aim: This work focus on the preclinical development of a new therapeutic approach based on a microemulsion (ME) as drug delivery system for dodecyl creatine ester (DCE). Materials & methods: DCE-ME was prepared by titration method. Novel object recognition (NOR) tests were performed before and after DCE-ME treatment on Slc6a8-/y mice. Results: Intranasal administration with DCE-ME improved NOR performance in Slc6a8-/y mice. Slc6a8-/y mice treated with DCE-ME had increased striatal ATP levels mainly in the striatum compared with vehicle-treated Slc6a8-/y mice which was associated with increased expression of synaptic markers. Conclusion: These results highlight the potential value of DCE-ME as promising therapy for creatine transporter deficiency.

Keywords: creatine; creatine transporter deficiency; dodecyl creatine ester; drug delivery; microemulsion; nasal administration; neurotherapeutics for CTD.

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Conflict of interest statement

Financial & competing interests disclosure

This work was supported by Fondation Le Jeune, Lucane Pharma and X-traordinaire (which is a patient group dedicated to intellectual deficiency rare diseases. Additional funding to MRS was provided by NIH (grant number: HD080910). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Figures

Figure 1.
Figure 1.. Rational design of dodecyl creatine ester-microemulsion strategy.
(A) Schematic representation of ME preparation. Solubilization of DCE in the inner lipid phase (Maisine®CC/DHA); surfactant addition (Transcutol® HP) under magnetic stirring; water titration to the previous mix and ME spontaneous formation; the final structure of the ME: a lipid inner phase in which DCE was previously solubilized surrounded by a surfactant shell and the external aqueous phase. (B) Ternary phase diagrams indicating o/w microemulsion region at different surfactant/oil/water mix ratios. Oil: Maisine®CC:DHA (1:1); oil: Maisine®CC:DHA (1:2). (C) Transmission electron microscopy micrograph of optimized DCE loaded ME. DCE: Dodecyl creatine ester; DHA: Docosahexaenoic acid; ME: Microemulsion.
Figure 1.
Figure 1.. Rational design of dodecyl creatine ester-microemulsion strategy.
(A) Schematic representation of ME preparation. Solubilization of DCE in the inner lipid phase (Maisine®CC/DHA); surfactant addition (Transcutol® HP) under magnetic stirring; water titration to the previous mix and ME spontaneous formation; the final structure of the ME: a lipid inner phase in which DCE was previously solubilized surrounded by a surfactant shell and the external aqueous phase. (B) Ternary phase diagrams indicating o/w microemulsion region at different surfactant/oil/water mix ratios. Oil: Maisine®CC:DHA (1:1); oil: Maisine®CC:DHA (1:2). (C) Transmission electron microscopy micrograph of optimized DCE loaded ME. DCE: Dodecyl creatine ester; DHA: Docosahexaenoic acid; ME: Microemulsion.
Figure 2.
Figure 2.. Improved object recognition memory in Slc6a8-/y mice and creatine content in different brain regions after intracerebroventricular treatment with dodecylcreatine ester.
(A) Novel object recognition tests were conducted 1 h after familiarization in Slc6a8-/y and Slc6a8+/y (n = eight animal/group) mice treated for 5 days with vehicle or DCE. The discrimination index was calculated as the difference between new and familiar object exploration times divided by total time spent observing both objects. Experimental comparisons with multiple groups were analyzed using a one-way ANOVA with the false discovery rate method of Benjamini, Kreiger and Yekutieli used for post hoc analysis. Data are mean ± standard error of the mean. (B) Creatine content in different brain regions of Slc6a8+/y and Slc6a8-/y mice (n = 4) after intracerebroventricular administration of DCE measured by LC-MS/MS. Data are mean ± standard error of the mean. Mann–Whitney test. *p ≤ 0.05. DCE: Dodecylcreatine ester; LC-MS/MS: Liquid chromatography tandem mass spectrometry.
Figure 2.
Figure 2.. Improved object recognition memory in Slc6a8-/y mice and creatine content in different brain regions after intracerebroventricular treatment with dodecylcreatine ester.
(A) Novel object recognition tests were conducted 1 h after familiarization in Slc6a8-/y and Slc6a8+/y (n = eight animal/group) mice treated for 5 days with vehicle or DCE. The discrimination index was calculated as the difference between new and familiar object exploration times divided by total time spent observing both objects. Experimental comparisons with multiple groups were analyzed using a one-way ANOVA with the false discovery rate method of Benjamini, Kreiger and Yekutieli used for post hoc analysis. Data are mean ± standard error of the mean. (B) Creatine content in different brain regions of Slc6a8+/y and Slc6a8-/y mice (n = 4) after intracerebroventricular administration of DCE measured by LC-MS/MS. Data are mean ± standard error of the mean. Mann–Whitney test. *p ≤ 0.05. DCE: Dodecylcreatine ester; LC-MS/MS: Liquid chromatography tandem mass spectrometry.
Figure 3.
Figure 3.. Improved object recognition memory in Slc6a8-/y mice after nasal administration of dodecylcreatine ester-microemulsion.
Novel object recognition tests were conducted 1 h after familiarization in Slc6a8-/y and Slc6a8+/y (n = eight animal/group) mice before and after 10 days of intranasal treatment with vehicle or DCE-ME. Data were analyzed using one-way ANOVA with the false discovery rate method of Benjamini, Kreiger and Yekutieli used for post hoc analysis. Data are mean ± standard error of the mean. *p ≤ 0.05. DCE-ME: Dodecylcreatine ester-microemulsion.
Figure 4.
Figure 4.. Nasal administration of dodecylcreatine ester-microemulsion rescue BDNF levels in the striatum of Scl6a8-/y mice.
(A) Real-time PCR shows that in the striatum of Scl6a8-/y mice, ME increases PSD95, CREB and BDNF levels compared with nontreated Scl6a8-/y mice. One-way ANOVA with the Tukey’s multiple comparison test for post hoc analysis were performed and *p < 0.001 indicates significant differences between nontreated Scl6a8-/y mice and treated Scl6a8-/y mice or between Scl6a8+/y mice and nontreated Scl6a8-/y mice. Data are represent mean ± standard error of the mean (n = 3–6). (B) Schematic representation of regulation of synaptic markers in the striatum brain region implicated in novelobject recognition memory. The increased expression of Psd95, Creb and Bdnf participate in the restoration of cognitive function in the creatine transporter deficiency mice model. *p < 0.001. DCE-ME: Dodecylcreatine ester-microemulsion.
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