Hyperpolarized Agents

Dynamic Nuclear Polarization (DNP)

The material to be polarized is dissolved in a glass-forming solvent (glycerol, ethanol or dimethylsulfoxide), or it can be used as it is if it forms a glassy structure when lowering the temperature. It is then doped with a stable radical species and then placed into the magnetic field, brought to very low temperature (1-2 K) and irradiated with a proper microwave frequency, at or near to the electron resonance frequency. Under these conditions, polarization transfer from electrons (whose polarization approaches 100% in this temperature range) to nuclei occurs via flip-flop transitions. The very unefficient nuclear relaxation in the solid ensures the maintenance of the nuclear spins alignment during the process. After the polarization transfer has taken place, the RF is switched off, the sample is raised above the liquid Helium level and is rapidly dissolved in hot buffer still inside the magnetic field. This dissolution method allows to rapidly reach the body temperature without significant loss of polarization. The sample is then injected into alive animals or added to cells suspensions for MRI/MRS acquisitions.

Para-Hydrogen Induced Polarization (PHIP)

The hydrogen molecule exists as two different nuclear spin isomers: ortho-hydrogen (o-H 2), corresponding to a triply degenerate state (S=1, 75% natural abundance), in which the two nuclear spins are parallel, and parahydrogen (p-H 2), corresponding to a singlet state (S=0, 25% abundance), in which the two nuclear spins are antiparallel. It is possible to enrich hydrogen in the para form at low temperature in the presence of a paramagnetic catalyst. The PHIP procedure is based on the chemical reaction of hydrogen enriched in the para form (commonly termed as para-hydrogen) with unsaturated substrates. The imbalance in the spin population of the para-hydrogen molecule is transferred to the product molecules, which result then transformed into hyperpolarized systems. In the hydrogenated molecule, polarization transfer to heteronuclei occurs via scalar couplings.

An hyperpolarized state is defined as a state in which the nuclear spin populations are altered with respect to the equilibrium value described by the Boltzmann equation. Since the signal intensity is proportional to the spin populations difference, hyperpolarization leads to an increase in the NMR signal intensity which may reach values as high as 105. This can be exploited in the fast acquisition of high resolution MR images and spectra and allows to use nuclei other than protons, allowing to obtain images which are characterized by high signal to noise (S/N) ratios and free from background noise because of the low natural abundance of heteronuclei such as 13C and 15N. With hyperpolarized contrast agents, images are acquired by the direct detection of the heteronucleus and the contrast is given by the difference in signal intensity between regions reached by the hyperpolarized 13C molecule and uninvolved zones. In this context, Dynamic Nuclear Polarization (DNP)and Para-Hydrogen Induced Polarization (PHIP)methods are currently under intense scrutiny for the preparation of 13C enriched hyperpolarized substances.

The DNP hyperpolarization procedure can be in principle applied to any molecule, provided that efficient methods for the rapid dissolution of the hyperpolarized substrate and the separation of the paramagnetic agent are available. Conversely, the use of the PHIP method requires hydrogenatable substrates, and implies the use of hydrogenation catalysts which must be removed before the in vivo administration. Its main advantage relies on the fact that it does not require the very low temperatures used in the DNP procedure, thus resulting in a simpler and cheaper methodology.

At CIM, the PHIP procedure is used to produce hyperpolarized molecules suitable as 13C MRI contrast agents.

Three different areas of research are currently developed:

  • Preparation of novel substrates for para-hydrogenation
  • Development of quick methods for the catalyst and organic solvent removal after para-hydrogenation
  • Development of procedures to convert the 13C anti-phase signal obtained after para-hydrogenation to in-phase signal (suitable for MRI)

1) Novel substrates for para-hydrogenation.

Substrates must satisfy the following requisites:

  • an unsaturated substrate is necessary (usually a triple bond containing molecule, that is efficiently para-hydrogenated in the presence of a suitable catalyst)
  • a 13C atom characterized by a long T 1value (which allows to cope with the polarization loss due to relaxation), must be adjacent to the unsaturation in order to allow polarization transfer from para-hydrogen through scalar coupling
  • The hydrogenation product must be water-soluble

Some examples of substrates which have been developed:

  • 13C-enriched unsaturated derivatives of glucose
  • 15N-enriched unsaturated derivatives of choline

Among the 13C hyperpolarized substrates that have been used for in-vivo studies, pyruvate is the most widely exploited for the investigation of pathologies. Since a de-hydrogenated precursor of pyruvate does not exist, a method named PHIP-SAH (PHIP-Side Arm Hydrogenation) has been developed in our laboratories that allows to obtain this HP metabolite through the following passages: 1) functionalization of the carboxylic group with an unsaturated alcohol (the Side-Arm); 2) hydrogenation of th e double-triple bond using parahydrogen; 3) spin order transfer from the parahydrogen protons to the 13C carboxylate nucleus (Magnetic Field Cycle); 4) cleavage of the hydrogenated side-arm by means of hydrolysis. Hydrogenation is carried out in an organic, hydrophobic solvent and the HP metabolite is extracted in the aqueous phase, according to the phase transfer method (see the following section). The PHIP-SAH strategy can be applied to other metabolites (e.g. lactate, acetate, ..) and, in principle, to all the molecules containing a carboxylic group.

p-Hydrogenation

2) Quick methods for the catalyst and organic solvent removal after para-hydrogenation

Para-hydrogenation reactions are usually carried out in organic solvent solutions, by using Rh cationic complexes as catalysts. Both the solvent and the catalyst are toxic and must be removed before injection of the HP product. This can be achieved by:

a) Ion exchange chromatography and spray distillation

b) Phase transfer

c) use of water-soluble catalysts and ion exchange chromatography

All the three possibilities are studied at the CIM.

The second methodology is of particular interest because it allows to carry out the para-hydrogenation in organic solvents (where its efficiency is higher), and then to remove catalyst, solvent and unreacted substrate in one step only by phase transfer, yielding ready-to-use pure water solutions of the HP product. It is possible to use a lypophilic unsaturated precursor, which is quickly converted after para-hydrogenation into a water-soluble compound of interest by addition of an aqueous phase, in which it is immediately extracted. By this route, hyperpolarized succinate has been produced by para-hydrogenation of maleic anhydride in chloroform and subsequent hydrolysis.

3. Transfer of parahydrogen spin order to 13C net polarization.

Spin order must be transferred from para-hydrogen protons to heteronuclei, in order to obtain heteronuclear net polarization that can be used for the acquisition of MR mages. This can be achieved by application of an opportune magnetic field cycle (MFC) or by the use of pulse sequences. The first method is currently exploited in the CIM laboratories. A dedicated set-up has been built by Aspect Imaging and it consists in a magnetic field shield (three concentric cylinders made of mu-metal) bearing a solenoid coil that is supplied with electric current controlled with a custom-written function. This allows to carry out MFC in a completely controlled manner.

p-Hydrogenation

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