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Fluorine-19 (19F) Labeled Benzothiazole Derivative as a Biosensor for detection of Alzheimer’s Disease using Magnetic Resonance Imaging
Yurii Shepelytskyi1, Michael G Campbell2,3, Francis T Hane2,3, Tao Li3, Vitalii Solomin4, Vira Grynko1, and Mitchell S Albert2,3,5

1Chemistry and Materials Science Program, Lakehead University, Thunder Bay, ON, Canada, 2Chemistry, Lakehead University, Thunder Bay, ON, Canada, 3Thunder Bay Regional Health Research Institute, Thunder Bay, ON, Canada, 4Enamine Ltd, Kyiv, Ukraine, 5Northern Ontario School of Medicine, Thunder Bay, ON, Canada

Synopsis

There are 5-7 million new cases of Alzheimer’s disease (AD) recorded each year worldwide. Typically, Positron Emission Tomography (PET) is used for the detection of amyloid b plaques allow for diagnosis of AD at early stages. However, PET has poor resolution in comparison to magnetic resonance imaging (MRI). The goal of the presented work was to demonstrate that an MR active derivative of the molecule Thioflavin-T, which contains 19F (2-[p-(Trifluoromethyl)phenyl]-1,3-benzothiazole (BTZ)), can be used to distinguish between samples of AD brains and healthy rat brains.

Introduction

Alzheimer’s disease (AD) is the most common cause of dementia with over 36.5 million related patients worldwide1. The main medical imaging targets are senile plaques composed of Aβ fibrils2, which potentially allows for diagnosis during the early stage of the disease. The most commonly used Aβ imaging technique is Positron Emission Tomography (PET)2. Despite the high sensitivity of PET, its special resolution is limited3,4 and significantly lower compared to MRI. For visualization of amyloid plaques using MRI, the desirable biosensor should have a high affinity to Aβ fibrils. 19F labeled molecules are attractive due to the high gyromagnetic ratio of 19F and significant signal-to-noise ratio (SNR) of detected signal. The objective of this study was to synthesize a 19F-containing ThT derivative 2-[p-(Trifluoromethyl)phenyl]-1,3-benzothiazole (BTZ) that can be detected at 3T and to demonstrate that this molecule could be used to distinguish between AD brains and healthy brains in the rat model.

Materials and Methods

The 2-[p-(Trifluoromethyl)phenyl]-1,3-benzothiazole (BTZ) was synthesized by reacting 99% 2-aminothiophenol (Sigma-Aldrich) with 98% 4-(Trifluoromethyl) benzoic acid (Sigma-Aldrich) in toluene in the presence of excess of PCl3 at 100°C. The structure of the synthesized molecule was confirmed by NMR using a Varian Unity INOVA 500 NMR spectrometer. To test the affinity of synthetized BTZ to Aβ fibrils, we conducted Surface Plasmon Resonance (SPR) measurements using a Nicoya (Waterloo, Canada) OpenSPR instrument. Phosphate buffer saline (PBS) was used as a running buffer. Five concentrations (1mM, 0.66 mM, 0.5 mM, 0.33 mM and 0.25 mM) of the BTZ in H2O-DMSO mixture were tested. To support the hypothesis that we can see a significant difference in MRI signal from BTZ in healthy and AD brains, three healthy (age of 17,17 and 19 months) and three (age of 12,28 and 11 months) Tg355F-AD rat brains were studied using clinical 3.0T MRI scanner equipped with a custom-built quadrature 19F coil. Magnetic resonance spectroscopy (MRS) experiments were conducted with complete hemispheres first, and then 1-2 mm slices of the hemispheres. To prepare brain samples for MRS, 1 ml of the 50 mM BTZ solution in methylene chloride was added, centrifuged, and then washed out twice with ethanol. Spectroscopy scans were acquired using the following parameters: TR/TE=6000/0.14ms, BW=32kHz, sampling number=2048, number of signal averages (NSA) = 25, 49, 81, 100 and 300. Measured MRS spectra were integrated from the -15 to +15 ppm to obtain the total amount of molecules absorbed by the brain tissue. The two-sample Welsh’s test was applied to values of integral to evaluate the difference between the AD and control brains.

Results

The structure of the obtained molecule was confirmed by NMR (Fig.1). An average value of SPR signal was calculated for five different concentrations of BTZ solution in the time range from 23.5 to 28.5 seconds (Fig.2). An average SPR signal as a function of concentration was fitted to the model hyperbolic function and the dissociation constant for complex amyloid fibrils-BTZ was obtained. It was equal to (3.95±0.07)10-4 M. The MRS spectra of the AD and Healthy hemispheres and cut brain tissue are shown in Fig. 3A (NSA = 300) and 3B (NSA = 100) respectively. The mean integral values of the AD and control brains spectra were equal to 2.41±0.46 and 2.04±0.69, respectively. The Welsh’s test showed the significance of this difference with p=0.045 (Fig.4).

Discussion

Value of the dissociation constant (KD) for complex amyloid fibrils-BTZ was equal to (3.95±0.07)10-4 M. This result is one order of magnitude lower than the published value for ThT molecule5. This was likely a result of the absence of an OH group in the benzothiazole part of the molecule. The affinity can be improved by additional modification of the biosensor. The peak at 0 ppm (Fig.3A) and peak at 0.26 ppm (Fig.3B) correspond to the white matter due to its lipophilic structure. Peaks at -2.4 (Fig.3A) and -1.8 ppm (Fig.3B) correspond to the gray matter. The origin of the peaks (maybe from Aβ plaques or soft muscle tissue, or both) at -1.43 (Fig.3A) and -1.04 (Fig.3B) ppm is unclear. It is clear that the intensity of this peak is much larger in an AD brain than in a healthy control brain, which makes it a potential molecular imaging biomarker for AD. The Welsh’s test proved the hypothesis that AD brain tissue absorbs a significantly higher amount of BTZ molecules than healthy brains (p=0.045) due to a high affinity of the synthetized molecule to the amyloid fibrils.

Conclusion

Overall, this study shows the potential of using the 19F labeled benzothiazole derivatives as an amyloid imaging biosensor for a clinical 3T scanner.

Acknowledgements

This work has been funded by an NSERC grant. Lakehead University and Thunder Bay Regional Health Research Institute provided support and access to their facilities. The authors acknowledge Michael Sorokopud for his experimental assistance with NMR spectrometer. The authors thank Alanna Wade for discussions of the MRS results. Francis Hane is supported by fellowships from the BrightFocus Foundation and the Canadian Institutes for Health Research. Yurii Shepelytskyi is supported by Ontario Graduate Scholarship. Vira Grynko is supported by Ontario Trillium Scholarship.

References

  1. Robinson, M., Lee, B. Y. & Hane, F. T. Recent Progress in Alzheimer’s Disease Research, Part 2: Genetics and Epidemiology. J. Alzheimers. Dis.2017; 57:317–330.
  2. Hane, F. T. et al. Recent Progress in Alzheimer’s Disease Research, Part 3: Diagnosis and Treatment. J. Alzheimer’s Dis. 2017; 57:645–665.
  3. Zhang, J., Maniawski, P. & Knopp, M. Effect of next generation SiPM digital photon counting PET technology on effective system spatial resolution. J. Nucl. Med. 2017; 58:1322.
  4. Cherry, S. R. et al. Total-Body PET: Maximizing Sensitivity to Create New Opportunities for Clinical Research and Patient Care. J. Nucl. Med. 2018; 59:3–12.
  5. Sulatskaya, A. I., Kuznetsova, I. M. & Turoverov, K. K. Interaction of thioflavin T with amyloid fibrils: Stoichiometry and affinity of dye binding, absorption spectra of bound dye. J. Phys. Chem. B. 2011; 155: 11519–11524.

Figures

Figure 1. The 1H (A) and 19F (B) spectra of synthetized BTZ. The white numbers indicate the integrals of the proton peaks.

Figure 2. A) The SPR binding curves of the five studied concentrations. B) The SPR signal dependence on the BTZ concentrations.

Figure 3. The MR spectra of AD and Healthy hemispheres (0.31 g and 0.38 g respectively) (A) and cut brain tissue (0.2 g) (B). The solid and dashed pointers show the peaks position in the AD and Control spectra respectively

Figure 4. Box chart of the integral value for all healthy and AD brains

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)
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