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Targeting the Endocannabinoid System for Neuroprotection: A (19)F-NMR Study of a Selective FAAH Inhibitor Binding with an Anandamide Carrier Protein, HSA.

Zhuang J, Yang DP, Tian X, Nikas SP, Sharma R, Guo JJ, Makriyannis A - J Pharm Pharmacol (Los Angel) (2013)

Bottom Line: We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds.Our results may provide insight into the mechanism of HSA-AM5206 interactions.The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA ; Department of Chemistry, The College of Staten Island, City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314, USA.

ABSTRACT
Fatty acid amide hydrolase (FAAH), the enzyme involved in the inactivation of the endocannabinoid anandamide (AEA), is being considered as a therapeutic target for analgesia and neuroprotection. We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds. In the present work, we characterized the interactions of AM5206 with a representative AEA carrier protein, human serum albumin (HSA), using (19)F nuclear magnetic resonance (NMR) spectroscopy. Our data showed that as a drug carrier, albumin can significantly enhance the solubility of AM5206 in aqueous environment. Through a series of titration and competitive binding experiments, we also identified that AM5206 primarily binds to two distinct sites within HSA. Our results may provide insight into the mechanism of HSA-AM5206 interactions. The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain.

No MeSH data available.


Related in: MedlinePlus

Integration of 19F-NMR signals from AM5206 in HEPES buffer with HSA (blue squares) and without HSA (red circles). The two horizontal axes represent the actual amount of AM5206 stock solution (50 mM in DMSO) added and the corresponding amount of AM5206 in the NMR sample (mg/mL). The vertical axis on the right shows the concentration of the soluble portion of AM5206.
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Figure 4: Integration of 19F-NMR signals from AM5206 in HEPES buffer with HSA (blue squares) and without HSA (red circles). The two horizontal axes represent the actual amount of AM5206 stock solution (50 mM in DMSO) added and the corresponding amount of AM5206 in the NMR sample (mg/mL). The vertical axis on the right shows the concentration of the soluble portion of AM5206.

Mentions: It is interesting to compare the integrations of the 19F-NMR spectra from these two parallel titration experiments. Since NMR detects only the “soluble” fraction of AM5206 in all sample preparations, it presents a unique methodology to quantify its solubility [33-35]. To ensure accurate integration, we first measured the integrals of different 19F-NMR spectra recorded using a range of recycle delays from 1 s to 30 s and confirmed that a 2 s recycle delay between scans is adequate. In Figure 4, we plotted the total integral values against the amount of AM5206 added in each NMR sample. When titrated into the HSA solution, the integral values increased linearly with the addition of AM5206. The black line represents the best-fit of the data points according to the linear regression model, and the r2 is found to be 0.993. Our data show that albumin can completely solubilize AM5206 up to 1.0 mg/ml concentration (30 μl AM5206 stock in 500 μl HSA solution). This corresponds to a 3.0 mM of AM5206 concentration in the HSA solution. In contrast, when AM5206 was added into HEPES buffer, the signal integral increased at the very beginning and leveled off for the remaining titration experiments. This corresponds to an AM5206 concentration of ~70 μM in the HEPES buffer solution. Therefore, albumin can significantly enhance the solubility of AM5206 in aqueous buffer solutions from ~70 μM to 3.0 mM, an approximately 50-fold increase.


Targeting the Endocannabinoid System for Neuroprotection: A (19)F-NMR Study of a Selective FAAH Inhibitor Binding with an Anandamide Carrier Protein, HSA.

Zhuang J, Yang DP, Tian X, Nikas SP, Sharma R, Guo JJ, Makriyannis A - J Pharm Pharmacol (Los Angel) (2013)

Integration of 19F-NMR signals from AM5206 in HEPES buffer with HSA (blue squares) and without HSA (red circles). The two horizontal axes represent the actual amount of AM5206 stock solution (50 mM in DMSO) added and the corresponding amount of AM5206 in the NMR sample (mg/mL). The vertical axis on the right shows the concentration of the soluble portion of AM5206.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921897&req=5

Figure 4: Integration of 19F-NMR signals from AM5206 in HEPES buffer with HSA (blue squares) and without HSA (red circles). The two horizontal axes represent the actual amount of AM5206 stock solution (50 mM in DMSO) added and the corresponding amount of AM5206 in the NMR sample (mg/mL). The vertical axis on the right shows the concentration of the soluble portion of AM5206.
Mentions: It is interesting to compare the integrations of the 19F-NMR spectra from these two parallel titration experiments. Since NMR detects only the “soluble” fraction of AM5206 in all sample preparations, it presents a unique methodology to quantify its solubility [33-35]. To ensure accurate integration, we first measured the integrals of different 19F-NMR spectra recorded using a range of recycle delays from 1 s to 30 s and confirmed that a 2 s recycle delay between scans is adequate. In Figure 4, we plotted the total integral values against the amount of AM5206 added in each NMR sample. When titrated into the HSA solution, the integral values increased linearly with the addition of AM5206. The black line represents the best-fit of the data points according to the linear regression model, and the r2 is found to be 0.993. Our data show that albumin can completely solubilize AM5206 up to 1.0 mg/ml concentration (30 μl AM5206 stock in 500 μl HSA solution). This corresponds to a 3.0 mM of AM5206 concentration in the HSA solution. In contrast, when AM5206 was added into HEPES buffer, the signal integral increased at the very beginning and leveled off for the remaining titration experiments. This corresponds to an AM5206 concentration of ~70 μM in the HEPES buffer solution. Therefore, albumin can significantly enhance the solubility of AM5206 in aqueous buffer solutions from ~70 μM to 3.0 mM, an approximately 50-fold increase.

Bottom Line: We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds.Our results may provide insight into the mechanism of HSA-AM5206 interactions.The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA ; Department of Chemistry, The College of Staten Island, City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314, USA.

ABSTRACT
Fatty acid amide hydrolase (FAAH), the enzyme involved in the inactivation of the endocannabinoid anandamide (AEA), is being considered as a therapeutic target for analgesia and neuroprotection. We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds. In the present work, we characterized the interactions of AM5206 with a representative AEA carrier protein, human serum albumin (HSA), using (19)F nuclear magnetic resonance (NMR) spectroscopy. Our data showed that as a drug carrier, albumin can significantly enhance the solubility of AM5206 in aqueous environment. Through a series of titration and competitive binding experiments, we also identified that AM5206 primarily binds to two distinct sites within HSA. Our results may provide insight into the mechanism of HSA-AM5206 interactions. The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain.

No MeSH data available.


Related in: MedlinePlus