Hyperpolarized ⁸⁹Y offers the potential of direct imaging of metal ions in biological systems by magnetic resonance

Abstract

Hyperpolarization of ⁸⁹YCl₃ and three ⁸⁹Y-complexes was achieved by dynamic nuclear polarization of aqueous samples. The long T₁’s of ⁸⁹Y make its application as an MR imaging probe extremely promising. In addition, the wide chemical shift range for various chelates of ⁸⁹Y means that agents sensitive to their biological/chemical milieu could serve as exquisite sensors of important biological events.

Hyperpolarization of nuclear spins can produce a dramatic increase in sensitivity for NMR active nuclei. Although the idea of transferring spin polarization from electrons to nuclei by dynamic nuclear polarization (DNP) to create a hyperpolarized NMR sample has been around since the mid-1950s, applications of this technology for study of liquid samples have appeared only recently. In 2003, Ardenkjaer-Larsen, et al.¹ developed an automated method to polarize ¹³C nuclei at low temperatures in the presence of a stable trityl radical then bring the sample to room temperature very quickly to perform NMR measurements.^1,2 Obviously, this method was most practical for long T₁ ¹³C nuclei such as non-protonated carbonyl or carboxyl carbons in rapidly tumbling small molecules which yield NMR signal enhancements of 10,000-fold or higher. One of the more exciting applications of this technology was reported shortly thereafter by Golman, et al.³ who demonstrated that it is practical to do real time metabolic imaging of [1-¹³C]pyruvate, [1-¹³C]lactate, and [1-¹³C]alanine in live animals using ¹³C chemical shift imaging (lactate and alanine are quickly formed from the injected hyperpolarized pyruvate through single enzyme catalyzed steps).

A commercial DNP device based on this technology now offers new opportunities for imaging nuclei that have not ordinarily been considered possible in the past. One attractive NMR nucleus for polarization is ⁸⁹Y because this nucleus is difficult to detect at thermal Boltzmann polarization levels due to its small magnetic moment, low receptivity and long T₁ relaxation times. ⁸⁹Y does however have a favourable spin quantum number (½), sharp NMR linewidths (3–5 Hz), and a long T₁ so this makes ⁸⁹Y attractive as a potential in vivo imaging and spectroscopy probe. Another isotope of yttrium, ⁹⁰Y (half life = 2.7 days), is an attractive radioisotope for cancer therapy because it emits high energy β electrons (average β energy = 0.93MeV) that provide relatively deep tissue penetration necessary for the treatment of larger tumors.^4,5 To date, the only approved targeted ⁹⁰Y radiopharmaceutical is Zevalin, used for treatment of non-Hodgkin's lymphomas.⁶ A second drug, ⁹⁰Y-DOTA-D-Phe¹-Tyr³-octreotide (⁹⁰Y-DOTATOC), is in phase I/II studies in Europe for somatostatin receptor positive tumors⁷ and others will likely follow. Thus, a long-lived, hyperpolarized ⁸⁹Y analog might be attractive for imaging the biodistribution of such drugs.

Only one ⁸⁹Y NMR study was reported prior to the 70’s.⁸ With the advent of FT spectrometers, a few more reports appeared demonstrating that ⁸⁹Y salts have unusually long T₁ values and are concentration dependent.⁹ More interestingly, Levy, et al.,¹⁰ were first to show that complexation of ⁸⁹Y³⁺ with crown ethers of various ring size resulted in lengthening of T₁ about 4-fold over that measured for salts dissolved in DMSO. In 1990, Holz and Horrocks used ⁸⁹Y³⁺ as a Ca²⁺ surrogate and reported that the chemical shift of various chelated forms of Y³⁺ varies widely, ranging from 36.6 ppm when bound at the EF site of parvalbumin to 129.6 ppm in Y(EDTA)⁻.¹¹ This was the first demonstration that the chemical shift of ⁸⁹Y³⁺ complexes could be used as a probe of the coordination environment of the ion. Given the potential of hyperpolarized ⁸⁹Y for molecular imaging of targeted therapeutics, we recently initiated DNP studies of a few Y³⁺ complexes using a commercial polarizer (HyperSense, Oxford Molecular Biotools).

Dynamic nuclear polarization of frozen solutions consisting of YCl₃ and three different chelated forms of Y³⁺ in the presence of a common stable trityl radical resulted in polarization enhancements that varied from 246 to 1527-fold above thermal equilibrium at 310K. Although these polarizations are small compared to recently reported ¹³C samples where polarizations of 5,000 – 10,000-fold have been achieved, they were sufficient to allow easy detection of the ⁸⁹Y NMR signal using a single 10° pulse (Figure 1). From an experimental standpoint, the necessity of reducing the sample volume by taking only a portion of the effluent from the dissolution process undoubtedly resulted in a loss of some magnetization due to T₁ processes before the NMR data collection ensued (see Supporting Information). Here, our goal was to measure T₁ values on each sample by following the decay process so the sample volume was limited to 1.5 mL to ensure that the entire volume was sampled with each 10° pulse. If the volume of the sample is not restricted to the sample coil, diffusion will cause erroneous estimates of the T₁ due to the cos^(n-1) term in equation 1 (Supporting information), making the T₁ appear artificially long. Also, the linewidth of the trityl radical used here is much too wide to match the Larmor frequency of ⁸⁹Y at the polarizing field (6.89 MHz) so other more favourable radicals should produce substantially higher polarizations. The polarization time used here (2.5 hours) was arbitrary and chosen simply for experimental convenience. In general, the DNP effect is mediated by the electron-nuclear dipolar interactions and the low gyromagnetic ratio of ⁸⁹Y means that the polarization time constant is likely very long. Thus, it is likely that higher polarization levels could have been achieved by polarizing the samples for a longer period.

Figure 1.

The polarization enhancements reported in Table 1 were determined by comparison of the intensity of the polarized ⁸⁹Y signal after the first 10° pulse to a 3M YCl₃ standard (single 90° pulse). Comparison of the signal from hyperpolarized samples after full relaxation (thermal polarization) was impossible at these concentrations (mM). The polarization predicted for a sample cooled to 1.4K is 221 times greater than the Boltzmann thermal value at 310K. The measured enhancements of YCl₃ and Y(DOTP)⁵⁻ were only slightly above this value so the effect due to DNP is only marginal in those cases. The remaining samples had polarizations well above that predicted for a cooled sample. It is interesting to note that the more highly charged species (Y³⁺ and Y(DOTP)⁵⁻) show the lowest polarization and, as the charge is reduced (Y(DTPA)²⁻ > Y(DOTA)⁻), polarization increases. This may reflect less than optimal glass formation at 1.4K with the more highly charged species.

Table 1.

Hyperpolarized ⁸⁹Y data for YCl₃ and Y^III complexes.

Compound	Conc. in NMR tube (mM)	Measured enhancement	Measured T1 (s)	Chemical shift (ppm)
YCl3	15	246	620	0 (ref)
Y(DTPA)2−	7.6	566	451	76
Y(DOTA)−	7.9	1527	499	109
Y(DOTP)5−	10	298	264	141
Y(DOTP)5−	5	1042	277	141

The T₁’s were measured by fitting the polarization decay curves (Figure 2) to equation 1 (Supporting Information). The T₁ values reported previously for Y(NO₃)₃ on thermally polarized samples varied from 63 – 270 s depending upon concentration (1M samples gave longer T₁ values than 3M samples).¹⁰ Our estimates of T₁ obtained by following the decay of hyperpolarized ⁸⁹Y are even longer than those reported by Levy, et al. by another factor of ~2.3 but the concentration of the hyperpolarized sample was also lower by a factor of ~67 so the trend reported by Levy, et al. appears to hold over a very wide concentration range. It is unclear however whether this is simply an effect due to concentration or whether it partially reflects the experimental difficulty in measuring such long T₁ values for such an insensitive nucleus. In general, the T₁’s of the ⁸⁹Y-chelates were found to be lower than that of the YCl₃ sample, also in contradiction to earlier results.¹⁰ One possible explanation for this observation is the higher B₀ field used here (14.1 Tesla) may have an additional chemical shift anisotropy contribution to the T₁ relaxation mechanism in the asymmetric environment of the chelates. It is also possible that other ligand spins in these samples (¹⁴N, ³¹P) contribute to the relaxation of ⁸⁹Y.

Figure 2.

NMR signal of ⁸⁹Y(DOTA)⁻ collected as a function of time after the sample was ejected from the polarizer, then inserted into the magnet. Total time following dissolution until the first acquisition was 30 seconds. The pH was 7 and final concentration of Y(DOTA)⁻ in the NMR tube was 7.9 mM. The fitted line gave a T₁ of 499 seconds.

In conclusion, hyperpolarization of ⁸⁹Y salts and ⁸⁹Y complexes is feasible with currently available commercial hardware. The long T₁’s of ⁸⁹Y make its application as an MR imaging probe quite promising. In addition, the wide chemical shift range for ⁸⁹Y means that contrast agents sensitive to a variety of biological/chemical milieu could serve as exquisite sensors of important biological events.

Supplementary Material

1si20070908_01. Supporting data available.

Experimental procedures as well as the equation used to fit the NMR data to obtain T₁. This material is available free of charge inline at http://pubs.acs.org.