d4.2 pk by combined cest : 13c nmr spectroscopy mk · title: microsoft word - d4.2 pk by combined...

D4.2 pK by combined CEST / 13C NMR spectroscopy

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“This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Grant Agreement No 667510“

Grant agreement no. 667510

GLINT

Research and Innovation Action H2020-PHC-2015-two-stage


Work Package: 4 Due date of deliverable: 31/12/2017

Actual submission date: 12/01/2018 Lead beneficiary: TAU

Contributors: TAU Reviewers: D. Longo (UNITO)

Project co-funded by the European Commission within the H2020 Programme (2014-2020)

Dissemination Level PU Public YES CO Confidential, only for members of the consortium (including the Commission Services)

CI Classified, as referred to in Commission Decision 2001/844/EC




Disclaimer The content of this deliverable does not reflect the official opinion of the European Union. Responsibility for the information and views expressed herein lies entirely with the author(s).




Contents 1 VERSION LOG ..................................................................................................... 4

2 INTRODUCTION .................................................................................................... 5

3 METHODOLOGY AND APPROACH .......................................................................... 6

4 RESULTS ............................................................................................................ 7

5 CONCLUSIONS .................................................................................................. 22

6 REFERENCES .................................................................................................... 23




1 Version log Version Date Released by Nature of Change

V1.0 19/12/2017 M. Rivlin First version

V1.1 12/01/2018 M. Kim Format changes, spell check




2 Introduction In order to separately assess glucose uptake and metabolism, both native and methylated glucose were measured together with a clear delineation of the in vivo characteristics of the metabolic pathway. The results obtained in here will allow to properly define the pK.




3 Methodology and Approach • 13C NMR experiments were performed on extracts from 4T1 Model (murine breast

tumours) and from murine brains following the administration of 13C labelled 3-O-

methyl glucose in order to assess the intracellular energy substrate levels.

• 31P NMR experiments were performed on extracts from 4T1 Model (murine breast

tumours) and from murine brains following the administration of 3-O-methyl glucose

in order to assess the intracellular energy substrate levels.

• Several methods of the administration of the agent (IV, IP, PO) were studied in

various animal models of solid tumours (4T1, MDA-MB-231, MCF7).

• Identification of the lowest detectable dose of 3OMG in breast cancer models.

• Measurements of the exchange rates of hydroxyl mobile protons belonging to glucose

and glucose derivatives as a function of the solution pH, PB concentration and at

different temperatures.

• Predicting in vivo equilibrium steady state of 3OMG solution to assure proper and

effective use of this innovative product by exploring the mutarotation activity of

3OMG.

• All CEST spectra presented here were acquired with a 500 MHz Bruker NMR

spectrometer with a range of saturation powers.

• Here we added data concerned with 13C and 31P NMR studies of extracts from murine

brains. The averaged alpha to beta anomeric ratio was 1 to 1.42, respectively.




4 Results Significant results achieved

• 13C NMR results indicate the penetration of 3OMG to tumours and brains, while no other

metabolic product could be observed.

• The 31P NMR spectra of extracts from 4T1 tumours also showed no evidence of any

phosphorylated products in the treated tumours and brains.

• Identification of the lowest detectable dose of 3OMG in breast cancer models- PO

administration of 570 mg/kg of 3OMG yielded 3-4% CEST above the baseline.

• Measurements the changes of the protons exchange rates of 3OMG as a function of pH

may serve to locate the 3OMG to the intra or extra cellular compartments.

• Accomplish a complete understanding of 3OMG mechanism by predicting in vivo

equilibrium of 3OMG solution.

• Data obtained by 13C and 31P NMR spectra of combined extracts from 4T1 tumors

following administration of [6-13C] 3OMG (1.0 g/kg, PO) was already shown in our

recent publication (1).

NMR spectroscopy 13C and 31P NMR were used to analyze 3OMG metabolism in murine brains. Mice were

administrated with [6-13C] 3OMG (1.0 g/kg, PO) and brains were excised within ~40min

after treatment. 13C and 31P NMR results for extracts of brains are shown in Figs.1 and 2. As

is seen from Fig. 1a-d, the 13C NMR of the extracts of brains points to a significant peak

(63.3ppm) originate from the administrated [6-13C]3OMG. This may serve as an indication to

the significant 3OMG CEST effect originates mainly from the intake of 3OMG into the

brains. The 13C NMR indicate that there are no other metabolic products besides 3OMG as it

is a non-metabolized glucose analog that enters the cells via the membrane concentrative

sodium dependent glucose transporter and exits the cells via the membrane facilitated

diffusional transporter.

Examination of 31P NMR difference spectra (Fig. 2) showed no formation or accumulation of

any glucose analog phosphate resonance after 3OMG administration, in the brains.




Figure 1: 1H-decoupled 13C NMR spectra of metabolites extracted from brains of mice bearing 4T1 model. a-d are extracts from brains of mice administrated with [6-13C] 3OMG (1.0 g/kg, PO). e is extract from control brain (without treatment). Each spectrum corresponds to an overnight data accumulation and represents a single specific brain of a mouse. The resonance of [6-13C] 3OMG is shown at 63.3 ppm in spectra a-d, respectively. The peaks were referenced to DSS (0 ppm).

Figure 2: 31P NMR spectra of metabolites extracted from brains of mice bearing 4T1 model. a and b are extracts from brains of mice administrated with [6-13C] 3OMG (1.0 g/kg, PO). c and d are extracts from control brains (without treatment). The peaks were referenced to GPC (0.49 ppm). Each spectrum represents a single specific




brain of a mouse. The peaks were assigned according to previously published data: GPC- glycerphosphocholine; GPE-glycerphosphoethanolamine; Pi- inorganic phosphate.

CEST measurments were assesed to follow the changes of the protons exchange rates of

3OMG as a function of pH. The results may serve to locate the 3OMG to the intra or extra

cellular compartments.

Assessing the intra/extracellular localization of 3OMG by measuring pH

The pH dependence of the MTRasym for 3OMG was published before at 25oC (Fig. 3) and

now we have results at 37oC (Fig. 4). The pH dependence of the MTRasym is not sensitive

enough to serve as a marker for the location of the 3OMG in the intracellular and

extracellular spaces. However, the protons exchange rate that can be assessed from the

dependence of the Z spectra with B1 rf field is expected to be much more sensitive to the pH.

For this goal we have now preliminary results of the exchange rates for both D-glucose and

for 3OMG solutions at T=37oC. Furthermore, we have measured the dependence of the

exchange rate of the phosphate buffer (PB) concentration. The analysis was done by the

algorithm that was based on the fit to Bloch McConnell (BM) equations that was supplied to

us by Dr. Moritz Zaiss (MPG). In the fitting we arbitrarily fixed the fractions fB and fD to two

and one hydroxyl groups at ~1.2 and ~3ppm from the water signal corresponding to 3.6E-04

and 1.8E-04 respectively for 20mM D-glu solution and 1.8E-04 and 0.9E-04 for 10mM

3OMG solution. The results reported here are preliminary and the fit was not very good.

However, this preliminary results show an indication of the monotonous trend of the

exchange rate at 1.2 ppm (kba), which increases by a factor of 1.44 with the increase of pH

from 6.48 to 7.37. We are currently continuing to work on the subject.




Figure 3: MTRasym plot of a 10 mM 3OMG solution (containing 10 mM phosphate buffer and 10% D2O) with pH values from 6.3–8 measured at different frequencies offset from water (B1 = 2.5 µT) (a) and at frequency offset of 1.2 ppm as a function of the rf saturation field (B1) (b). (T= 25°C).

Figure 4: MTRasym plot of a 10 mM 3OMG solution (containing 10 mM phosphate buffer and 10% D2O) with pH values from 6.34–8.02 measured at different frequencies offset from water (B1 = 2.5 µT) (a) and at frequency offset of 1.2 ppm as a function of the rf saturation field (B1) (b). (T= 37°C)




I. CEST quantification for a solution of 10mM 3OMG, pH=6.49-7.37, 10mM PB,

10% D2O, T=37oC

Figure 5: Z spectra with BM fit to 10mM 3OMG solution, 10mM PB, 10%D2O, pH=6.49, T=37oC

Figure 6: MTRasym plot of 10mM 3OMG solution, 10mM PB, 10%D2O, pH=6.49, T=37oC





Figure 11: Bar graph showing the % of MTRasym of 10mM 3OMG solution (10% D2O) at pH=6.49 at

frequencies offset of 1.2, 2.1 and 2.9 ppm form the water signal, at T=37oC








Figure 14: The exchange rates of two hydroxyl metabolites (~1.2 and ~3ppm from the water signal) of 10mM

3OMG solution at different pH values

In order to see whether the proton exchange rate of D-Glu depends on the environment

conditions, we tested its dependence on the phosphate buffer concentration.




II. CEST quantification for a solution of 20mM D-glucose, pH=7.4, 0-50mM PB,

10% D2O, T=37oC

Figure 15: Z spectra with BM fit to 20mM D-Glu solution, 0mM PB, 10%D2O, pH=7.4, T=37oC

Figure 16: MTRasym plot of 20mM D-Glu solution, 0mM PB, 10%D2O, pH=7.4, T=37oC





Figure 27: Bar graph showing the % of MTRasym of 20mM D-Glu solution (10% D2O) at different PB

concentrations at frequency offset of 1.2 ppm form the water signal, at T=37oC




Figure 30: The exchange rates of two hydroxyl metabolites (~1.2 and ~3ppm from the water signal) of 20mM

D-Glu solution at different PB concentrations

Following the mutarotation reaction of the anomeric protons of deuterated 3OMG allowed to

obtained the kinetics constants of 3OMG. In the temperature range of 4-50oC the averaged

alpha to beta anomeric ratio was 1 to 1.42, respectively.




5 Conclusions • 13C NMR of extracts of brains of mice following administration of [6-13C] 3OMG (1.0

g/kg, PO) indicated the penetration of 3OMG to the brains, and no other metabolic

product could be observed. This is corroborating the generally excepted 3OMG as "non-

metabolizabled" glucose analogue.

• 31P NMR indicated no change in the metabolic profile of the brains upon the penetration

of 3OMG.

• The changes of the protons exchange rates of 3OMG as a function of pH give us the hope

that by the measurements of these rates we will be able to locate the 3OMG to the intra or

extra cellular compartments. However, the dependence of the exchange rate on the PB

concentrations will make this assignment less straightforward. In parallel, we started to

investigate this problem by different route, i.e. by the modification of the MTRasym

intensities by paramagnetic gadolinium complexes which reside exclusively at the extra

cellular compartments.

• The anomeric equilibrium constant and the rate of mutarotation of 3OMG were measured

by 13C and 1H high resolution NMR. At physiological conditions the mutarotation process

is very slow and is fully completed within a few hours. This property should be

considered when planning in vivo measurements involving the use of 3OMG and in the

calculation of proton exchange rates from CEST studies.




6 References 1. Rivlin, M. and Navon, G. (2017), CEST MRI of 3-O-methyl-D-glucose on different

breast cancer models. Magn. Reson. Med. doi:10.1002/mrm.26752

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