PREPARATION OF SOLID AMORPHOUS SUBSTRATES FOR DNP
20170252464 · 2017-09-07
Assignee
Inventors
- Elena ELISEI (Trieste, IT)
- Attilio CESARO (Trieste, IT)
- Marta FILIBIAN (Pavia, IT)
- Pietro CARRETTA (Pavia, IT)
- Sonia COLOMBO SERRA (Vigliano Biellese, IT)
- Alessandro Maiocchi (Monza, IT)
- Fabio TEDOLDI (Marzano, IT)
Cpc classification
International classification
Abstract
A method for preparing a sample for dynamic nuclear polarization which comprises submitting a solid substrate to a milling process in the presence of a polarizing agent at a temperature lower than the glass-transition temperature (Tg) of said substrate. Preferably said substrate may be in crystalline form.
Claims
1. A method for the manufacture of a sample in amorphous form for dynamic nuclear polarization which comprises submitting a solid substrate to a milling process in the presence of a polarizing agent at a temperature lower than the glass-transition temperature (Tg) of said substrate.
2. The method according to claim 1 wherein said solid substrate is in a crystalline form.
3. The method according to claim 1, wherein said substrate is selected from the group consisting of: amino acids, carbohydrates, hydroxy-acids, dicarboxylic acids and ketoacids.
4. The method according to claim 1, wherein said substrate is selected from the group consisting of arginine, glutamine, glucose, lactose, trehalose, lactic acid, malic acid and succinic acid.
5. The method according to claim 1, wherein said substrate is selected from the group consisting of D-(+)-Glucose, α-Lactose and Trehalose β-polymotph.
6. The method according to claim 1, wherein two or more substrates are in solid crystalline form and they are mixed with the at least one polarizing agent.
7. The method according to claim 1, wherein said at least one polarizing agent is an organic free radical.
8. The method according to claim 7 wherein said radical is selected from the group consisting of porphyrexide, TEMPO, TEMPONE, TEMPOL, tris(8-carboxy-2,2,6,6-(tetra(hydroxyethyl)-benzo-[1,2-4,5′]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt, trist8-carboxyl-2,2,6,6-tetramethyl-benzo(1,2-d:4,5-dS)bis(1,3)dithiole-4-yl)methyl sodium salt and 1,3-bisdiphenylene-2 -phenylallyl (BDPA).
9. The method according to claim 1, wherein the polarizing agent concentration on the total mixture weight is between 0.1% and 1.5%;
10. The method according to claim 1 wherein said milling is performed at a temperature at least 10° C. lower than the glass-transition temperature of said substrate.
11. The method according to claim 1 wherein said at least one substrate has a glass-transition temperature (Tg) above room temperature and said milling is performed at room temperature.
12. The method according to claim 1 wherein said milling is performed using a planetary ball mill.
13. A method for preparing a polarized sample comprising a substrate polarized by dynamic nuclear polarization (DNP) which comprises the following steps: submitting the solid substrate to a milling process in the presence of a polarizing agent according o the method of claim 1 thus obtaining a sample for DNP in amorphous form; b) submitting said sample to DNP to obtain a polarized sample comprising said polarized substrate.
14. A method for preparing a solution for MR analysis comprising the following steps: a) preparing a polarized sample using the method of claim 13, and b) dissolving said polarized sample in an appropriate solvent.
15. A method of MR analysis comprising (a) administering a solution for MR analysis comprising a polarized substrate prepared according to the method of claim 14 to a subject and (b) detecting a MR signal from said polarized substrate and/or from a metabolic product thereof.
16. The method according to claim 7 wherein said at least one polarizing agent is selected from the group consisting of: triarylmethyl radicals, nitroxide radicals, nitrogen centred radicals, oxygen centred radicals, stable carbon centred, radicals and mixtures thereof.
17. The method according to claim 1 wherein the, polarizing agent concentration on the total mixture weight is between 0.2% and 0.8% (w/w).
18. The method according to claim 1 wherein said milling is performed at a temperature at least 30° C. lower than the glass-transition temperature of said substrate.
19. The method according to claim 1 wherein said milling is performed at a temperature at least 50° C. lower than the glass-transition temperature of said substrate.
Description
FIGURES
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DEFINITIONS
[0063] Within the scope of the present invention, the terms “hyperpolarization”, “hyperpolarized” or similar mean enhancing the nuclear polarization of NMR active nuclei present in the target agent as commonly intended in the state of the art. The term “hyperpolarized” refers in particular to the nuclear spin polarization of a compound higher than thermal equilibrium.
[0064] As used herein, the term Dynamic Nuclear Polarization (DNP) refers to a technique for (hyper)polarizing a substrate, which comprises submitting the substrate in the presence of a polarizing agent to a microwave irradiation at very low temperatures as commonly intended in the state of the art and for example disclosed in WO200977575 and the references cited therein.
[0065] Within the scope of the present invention, the term “Magnetic Resonance investigation techniques”, includes within its meaning any diagnostic technique which comprises administration of a (hyper)polarized substrate and the detection of MR signals of said substrate or of a metabolite thereof as commonly intended in the state of the art, including in vivo imaging metabolic activity, as disclosed for example in WO200977575 and the references cited therein.
[0066] Within the meaning of the present invention, the term “substrate” includes any compound enriched with non-zero nuclear spin nuclei (such as .sup.1H, .sup.13C, .sup.19F and/or .sup.15N nuclei) which, when submitted to a DNP process, is capable of enhancing the nuclear polarization of its NMR active nuclei, i.e. “polarizes”. The polarized substrate is in particular suitable for use in subsequent MR investigation techniques; preferably the substrate, e.g. when administered in vivo, is capable of being converted into respective polarized metabolic compound(s).
[0067] As used herein, the term “solid”, when referred in particular to the substrate, includes within its meanings substances which are typically solid at room temperature, i.e. between 20° C. and 25° C. Within the meaning of the present invention, the term “milling” means the process of breaking down materials, in particular using a mill, more in particular a ball mill. “Grinding” is also herein used as a synonym of milling.
[0068] Within the meaning of the present invention, the terms “polarizing agent” and “paramagnetic agent” are used as synonyms.
[0069] Within the meaning of the present invention, the terms “polarized” and “hyperpolarized” are used as synonyms.
DETAILED DESCRIPTION OF THE INVENTION
[0070] The method of the invention can be used when the polarization of a solid substrate is required.
[0071] The method of the present invention can be applied in particular to any solid substrate having a crystalline form.
[0072] Preferably, it is applied to crystalline substrates stabilized by weak chemical bonds, such as Van der Waals, polar and/or H-bond interactions.
[0073] In a preferred embodiment, said substrate is an organic molecule having a crystalline form stabilized by polar and/or H-bond interactions.
[0074] In a more preferred embodiment, said substrate is selected from the group consisting of: amino acids, carbohydrates, hydroxy-acids, dicarboxylic acids and ketoacids.
[0075] When said substrate is an amino acid, it is preferably selected from arginine and glutamine. When said substrate is a carbohydrate, it is preferably selected from glucose, lactose and trehalose. When said substrate is an hydroxy-acid, it is preferably selected from lactic acid and malic acid. When said substrate is a dicarboxylic acid, it is preferably succinic acid.
[0076] Preferred solid compounds to be used as substrates are D-(+)-Glucose, α-Lactose and Trehalose β-polymorph.
[0077] The method of the invention may also be used for milling more than one solid substrate with the radical(s), in order to obtain a mixture of solid substrates and radical(s) to use as a single sample for DNP.
[0078] This is particularly useful when a single formulation containing a cocktail of substrates is desired, for example to simultaneously probe the activity of different enzymes.
[0079] For example, a mixture of crystalline lactose and crystalline glucose can be milled together with the selected radical according to the method of the invention to obtain a single amorphous sample to polarize.
[0080] According to the method of the invention, the solid substrate is milled together with a polarizing agent. Said polarizing agent is preferably a paramagnetic organic free radical. The polarizing agent is preferably solid at room temperature.
[0081] Examples of suitable polarizing agents are disclosed, for instance, in U.S. Pat. No. 6,311,086 and include triarylmethyl radicals, nitroxide radicals, nitrogen centred radicals, oxygen centred radicals, stable carbon centred radicals and mixtures thereof.
[0082] Preferred organic free radicals are nitroxide radicals (such as porphyrexide, TEMPO, TEMPONE and TEMPOL), and stable carbon centered radicals (such as trityls or allyls radicals).
[0083] Examples of preferred radical compounds are 2,2,6,6,-Tetramethyl-1-piperidinyloxy (TEMPO), TEMPONE, TEMPOL, (tris(8-carboxy-2,2,6,6-(tetra(hydroxyethyl)-benzo-[1,2-4,5′]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt or tris(8-carboxyl-2,2,6,6-tetramethyl-benzo(1,2-d:4,5-dS)bis(1,3)dithiole-4-yl)methyl sodium salt and 1,3-bisdiphenylene-2-phenylallyl (BDPA).
[0084] A particularly preferred radical is TEMPO.
[0085] The polarizing agent concentration on the total mixture weight can be up to 2% (w/w); preferably it is from 0.1% to 1.5%, more preferably from 0.2% to 0.8% (w/w).
[0086] Preferably, the total volume of the mixture comprising the substrate and the radical, typically contained in a milling jar of a total volume of 45 ml, is of from 0.4 to 35 ml, more preferably from 0.6 to 15 ml and even more preferably from 0.7 to 3 ml. This allows to obtain an optimal homogeneous amorphization and a good mixing of the sample and the radical.
[0087] When the substrate is a crystalline solid, the polarizing agent may be admixed together with the substrate at any time during the milling process, e.g. before, during or at the end of the milling process (i.e. when the sample has reached the desired amorphous state). In this latter case, the amorphized sample can optionally be stored as such before admixing it with the polarizing agent. The polarizing agent is then admixed with the amorphized sample and the mixture is submitted to a co-milling process, in order to reach the desired intimate and homogeneous dispersion of the polarizing agent into the amorphous sample. Optionally, a partially amorphous substrate can be first obtained, which is then admixed with the polarizing agent and the mixture is then submitted to co-milling in order to obtain the desired amorphous sample with the polarizing agent intimately and uniformly dispersed therein.
[0088] According to a preferred embodiment, the polarizing agent is first admixed with the substrate and the mixture is then submitted to a co-milling process.
[0089] As already explained above, the method of the invention can be carried out at room temperature for those substrates having a glass-transition temperature (Tg) above room temperature (preferably at least 10° C. higher than room temperature, more preferably at least 30° C. higher and even more preferably at least 50° C. higher than RT).
[0090] For substrates having a Tg close to or lower than RT, the mill is preferably equipped with a cooling system, in order to perform the milling process at the required temperature lower than said Tg, preferably of at least 10° C. lower than Tg, more preferably of at least 30° C. lower than the Tg and even more preferably 50° C. lower than the Tg.
[0091] The glass transition of a material is a pseudo second-order phase transition occurring during a cooling process of a melt or liquid-like state (undercooled liquid) that becomes a glass (amorphous solid), provided that the cooling rate is fast enough that crystallization is avoided. The glass transition may typically be observed through calorimetric or thermal analysis techniques in the cooling mode as well as, once the glass is formed, in the heating mode from glass to undercooled liquid. The glass transition temperature of a material (Tg) may vary slightly depending from the choice of the measurement technique and from the applied parameters (e.g. heating rate in Differential Scanning calorimetry (DSC)). In accordance with common practice, where not otherwise specified herein, in the present specification and claims the glass transition is determined by DSC with heating scanning rate of 5° C./min and the value of Tg is taken as the midpoint of the quasi-sigmoidal portion of the DSC curve corresponding to the increase of specific heat (C.sub.p) occurring at the glass transition.
[0092] Milling can be performed with any suitable instrument providing sufficient mechanical energy to disrupt the crystalline lattice of the substrate.
[0093] Preferably, the milling is performed using a ball mill, more preferably a planetary ball mill, even more preferably a high-energy planetary ball mill.
[0094] As an example, U.S. Pat. No. 6,126,097 discloses a high-energy planetary ball milling apparatus and protocol suitable for carrying out the method of the invention.
[0095] A suitable commercially available high-energy planetary ball milling apparatus is, for instance, Pulverisette 7 classic line (Fritsch, Germany).
[0096] In an embodiment of the invention, particularly when the substrate is in crystalline form, milling is performed for a time suitable to obtain a sample in the amorphous state. Preferably, it is performed for a time comprised between 1 hour and 24 hour. More preferably it is performed for a time comprised between 6 hours and 12 hours. The suitable time can be chosen by the skilled in the art depending on the compound to be milled and on the temperature at which milling is performed. These milling times are generally sufficient to allow an intimate an homogeneous mixing of the polarizing agent admixed with the substrate.
[0097] In another embodiment of the invention, particularly when the substrate is an amorphous or (partially) amorphized solid (e.g. obtained from a first milling step), milling is performed for a time suitable to obtain a homogeneous dispersion of the radical into the solid substrate. Preferably, it is performed for a time ranging between 10 minutes and 24 hours. More preferably it is performed for a time ranging between 1 hour and 6 hours.
[0098] While the above parameters and ranges are provided with respect to laboratory or pilot scale processes, based on the general knowledge and on the specific disclosure of the present invention, the skilled person is able to easily adapt these parameters and ranges to respective semi-industrial or industrial scale processes.
[0099] The achievement of an amorphous state—i.e. the substantial absence of crystalline phases in the sample—can be determined by conventional techniques such as, for instance, Raman spectroscopy and DSC. A Raman spectrum characteristic of a crystalline state presents many narrow and intense peaks (9), while a spectrum characteristic of an amorphous state presents very few broad and less intense bands. A calorimetric thermogram characteristic of a crystalline state presents only one (for one molecular species) exothermic narrow peak at the melting temperature (T.sub.m); on the other hand, the thermogram of an amorphous state presents an exothermic broad band of absorbed water evaporation at T<100° C. (for scan rate=5° C./min), a quasi-sigmoidal change in the specific heat C.sub.p at the glass transition temperature Tg (transition from glass to “undercooled liquid”), an endothermic peak underlining the re-crystallization and an exothermic peak at T, underlining the melting of the recrystallized phase.
[0100] As previously mentioned, the present process not only allows obtaining the substrate in the desired amorphous form but also allows an intimate and uniform dispersion of the polarizing agent (in particular of the radical) in the amorphous matrix of the sample.
[0101] In view of the above, the method of the invention provides a sample particularly suitable for an efficient polarization.
[0102] The obtained sample has also the advantage of remaining stable in the amorphous state for a relatively long period of time.
[0103] Therefore, it can be stored in order to preserve the amorphous state over sufficiently long time periods. This is particularly important for use of the amorphous sample for pharmaceutical applications.
[0104] To preserve the amorphous state, the obtained sample is preferably be maintained below its glass-transition temperature, preferably at a temperature of 10° C. lower than the Tg, more preferably at a temperature of 50° C. lower than its Tg.
[0105] Moreover, it can be preferably maintained in a controlled atmosphere, in particular in low humidity conditions, to further preserve its stability over longer periods of time.
[0106] For instance, the milled sample may remain stable (i.e. substantially amorphous) for at least 20 days under air or for at least 72 days under inert atmosphere (e.g. argon, O.sub.2 and H.sub.2O content<0.1 ppm).
[0107] The amorphous sample obtained by milling the substrate with the radical according to the method of the invention can then be hyperpolarized according to standard dynamic nuclear polarization protocols.
[0108] Reference can be made for example to WO98/58272 or WO2011124672.
[0109] An efficient DNP process is usually performed at high magnetic field (for example 3-8 T) and low temperatures (for example 0.5-5 K).
[0110] A polarized (DNP) sample comprising the polarized substrate of interest is thus obtained.
[0111] After the DNP process, the sample can be dissolved in an appropriate solvent, for example water, or solvent mixtures in order to obtain a solution for Magnetic Resonance Imaging (MRI) analysis.
[0112] A further aspect of the invention relates therefore to a method for preparing a solution for MRI analysis which comprises the steps of: [0113] a) preparing a solid polarized sample as above described, and [0114] b) dissolving the solid polarized sample in an appropriate solvent.
[0115] Dissolution of the sample is performed by addition of a suitable dissolving medium, preferably a physiologically tolerable aqueous carrier. Preferably the dissolution is performed in a relatively short time (few seconds) while the sample is maintained in the high magnetic field, for instance by admixture with hot aqueous carrier. Details on the dissolution process of a frozen hyperpolarised mixture and suitable devices for performing the dissolution are provided, for instance, in WO-A-02/37132.
[0116] Substances commonly used to regulate the pH at physiological values (e.g. HCl, NaOH, buffers) can also be added to the solution to obtain a sample suitable for injection in a subject. The appropriate substances should be chosen according to the component(s) of the solid sample and this can be done by the skilled person referring to its general knowledge in the field.
[0117] The radical and or reaction products thereof may be removed from the dissolved polarized compound. Methods usable to partially, substantially or completely remove the radical and/or reaction products thereof are known in the art. Generally, the methods applicable depend on the nature of the radical and/or its reaction products. For example, upon dissolution of the solid hyperpolarized compound, the radical might precipitate and then be easily separated from the liquid by filtration. If no precipitation occurs, the radical may be removed by chromatographic separation techniques, e.g. liquid phase chromatography like reversed phase, straight phase or ion exchange chromatography or by extraction. Alternatively, free radicals can be eliminated by adding a compound acting as a “scavenger” (for example a reducing agent like sodium ascorbate) to the mixture of substrate with radical so that when the mixture is dissolved said “scavenger” neutralizes the radical (see for reference U.S. Pat. No. 8,564,288B2).
[0118] The solution containing the hyperpolarized sample obtained with the method of the invention can thus be used as an MRI contrast agent for both in vitro and in vivo applications.
[0119] For example, it can be used in vitro on isolated biological samples or on cell cultures or administered to an animal or human subject for in vivo applications. Typically the solution containing the hyperpolarized sample is administered (e.g. by injection) within few seconds from the preparation thereof. In general, optimal concentrations will in most cases lie in the range from 10 mM to 250 mM, particularly from 150 to 250 mM. In any case, the dosage of the solution should be kept as low as possible whilst still providing a detectable signal of the polarized substrate or metabolites thereof.
[0120] The MR signals detected from the hyperpolarized substrate(s) or metabolites thereof can be used to generate an image, physiological data or metabolic data.
[0121] Indeed, it is a further object of the present invention a method of MR analysis comprising administering to a subject a solution for MR analysis comprising an hyperpolarized sample prepared as above described and detecting the resulting MR signal.
[0122] MR analysis can be performed according to standard protocols known in the art.
[0123] The method of the invention is preferably used for obtaining hyperpolarized samples of organic substrates which can be used as tracers for metabolic applications, for example to follow the metabolic conversion of a certain organic compound or for the detection of metabolic abnormalities associated with diseases.
[0124] The present invention will be further illustrated by the following non-limiting examples.
EXAMPLES
[0125] Where not otherwise specified, chemicals and reagents used in the following examples are commercially available or can be prepared according to methods well-known in the art.
[0126] Materials
[0127] The following materials have been employed in the subsequent examples: [0128] commercial crystalline TEMPO (2,2,6,6,-Tetramethyl-1-piperidinyloxy, from Sigma Aldrich) as solid radical component; [0129] commercial anhydrous crystalline β-Trehalose (from Acros Organics); [0130] commercial anhydrous crystalline α-Lactose (from Acros Organics); [0131] commercial crystalline D-(+)-Glucose (from Sigma-Aldrich); [0132] commercial crystalline Fumaric Acid (from Sigma-Aldrich).
[0133] Methods
[0134] The sample preparation is made by admixing the substrate to be polarized with the polarizing agent (TEMPO, 2,2,6,6,-Tetramethyl-1-piperidinyloxy) and submitting the mixture to milling by using a high energy planetary micro mill (Pulverisette 7 classic line from Fritsch, Germany). Each sample has been inserted in a ZrO.sub.2 milling bowl (volume about 45 cm.sup.3) containing seven ZrO.sub.2 balls (diameter 15 mm) and the rotation frequency of the solar disk has been set at 400 rpm. A homogeneous amorphization and a good mixing of the two species is reached by using a total mass of at least 1.1 g and a mixing time of at least 12 hrs (7, 8, see also U.S. Pat. No. 6,126,097). The process temperature was kept always lower than the Tg (T.sub.process<T.sub.g of 50° C.); pauses in the process were inserted to avoid raising in temperature (5 minutes every 15 minutes of milling at 400rpm). The achievement of an amorphous state—i.e. the absence of crystalline phases—was determined by Raman spectroscopy and DSC. A Raman spectrum characteristic of a crystalline state presents many narrow and intense peaks (9), while that characteristic of an amorphous state presents very few broad and less intense bands. A calorimetric thermogram characteristic of a crystalline state presents only one (for one molecular species) exothermic narrow peak at the melting temperature (T); on the other hand, the thermogram of an amorphous state presents an exothermic broad band of absorbed water evaporation at T<100° C. (for scan rate=5° C./min), a quasi-sigmoidal change in C.sub.p at the glass transition temperature T.sub.g (transition from glass to “undercooled liquid”), an endothermic peak underlining the re-crystallization and an exothermic peak at T.sub.m underlining the melting of the recrystallized phase.
[0135] Aliquots of milled sample have been used for the characterization of the structure and the analysis of the polarization properties: [0136] about 100 mg for Raman Spectrometric measurements; [0137] about 5-10 mg for Differential Scanning calorimetric measurements; [0138] about 5 mg for Thermogravimetric measurements; [0139] about 65 to 85 mg for Dynamic Nuclear Polarization measurements.
Example 1
Preparation and Characterization of a Milled Trehalose Sample for DNP
Raman and Calorimetric Measurements on a Crystalline (β) Trehalose
[0140] Two aliquots of crystalline β-Trehalose have been analyzed respectively by means of Raman Spectroscopy and Differential Scanning calorimetry, giving the results in
[0141] Preparation and Characterization of Milled Trehalose Sample for DNP
[0142] A mixture of crystalline Trehalose and crystalline TEMPO has been co-milled for 12 h at room temperature. In particular samples with radical concentration in the mixture of 0.15, 0.34, 0.50, 0.64 and 0.81% (w/w) have been prepared.
[0143] The milled sample with 0.50% of TEMPO has been analysed by means of Raman Spectroscopy and Differential Scanning calorimetry, giving the results in
Example 2
Preparation and Characterization of a Milled Lactose Sample for DNP
[0144] A mixture of crystalline Lactose and crystalline TEMPO has been co-milled for 12 h at room temperature; the radical concentration in the mixture is 0.48% (w/w).
[0145] The resulting sample, analyzed by means of Differential Scanning calorimetry (
Example 3
Preparation and Characterization of a Co-Milled Lactose-Glucose Sample for DNP
[0146] A mixture of crystalline Lactose, crystalline Glucose and crystalline TEMPO has been co-milled for 12 h at 12° C.; the radical concentration in the mixture is 0.60% (w/w) while the ratio between the Glucose and Lactose masses is 1:4.
[0147] The resulting sample, analyzed by means of Differential Scanning calorimetry (
Example 4
Preparation of a Milled Fumaric Acid Sample for DNP
[0148] A mixture of crystalline Fumaric Acid and crystalline TEMPO has been co-milled for 12 h at 12° C.; the radical concentration in the mixture is 0.49% (w/w).
Example 5
Thermogravimetric Measurements on Milled Lactose
[0149] An aliquot of the milled sample of Lactose (see Example 2) has been analyzed during the 2nd day after the amorphization by means of thermogravimetry.
[0150] Similar behaviour is observed for the other milled samples composed by sugars, while the milled Fumaric Acid is expected to contain a smaller amount of water.
Example 6
DNP Enhancement of Milled Trehalose as a Function of the Magnetic Field
[0151] An aliquot of the milled sample of Trehalose with radical concentration of 0.64% prepared according to Example 1 has been transferred into a quartz tube inside a glove box at room temperature and the tube has been capped by using Teflon tape. The sample has been cooled in a bath cryostat at 1.7 K and irradiated with MicroWaves (MW) at 96.93 GHz with a 30 mW power source. The 1H NMR signal enhancement (ε, i.e. the ratio between the steady state signal intensity under MW irradiation and the steady state signal intensity at thermal equilibrium) has been measured upon varying the applied magnetic field H between 3.44 T and 3.475 T. The behaviour of the polarization enhancement (ε) versus the applied magnetic field (H) has a shape that recalls the derivative of the EPR line of the TEMPO radical as a function of the magnetic field (
Example 7
DNP Enhancement of Milled Trehalose as a Function of Radical Concentration
[0152] The MW irradiation frequency was fixed to the 96.93 MHz to maximize the positive enhancement of the NaAc reference at 3.46 T. The milled samples of Trehalose (see Example 1) were transferred in quartz tubes (the procedure is described in the Example 6). Then ε was measured in all the samples between 4.2 K and 1.7 K. The data in
Example 8
Maximum Achievable DNP Polarization (P) of Several Milled Samples
[0153] On the first day after the amorphization, 50-70 mg of the milled sample of Trehalose and TEMPO (0.64%) and of the samples described in the Examples 2-4 were transferred in quartz tubes according to the procedure described in Example 6. A tube containing Lactose (sample 1) was immediately inserted in the helium bath for DNP measurements while the other tubes were stored in the freezer for subsequent measurements on days indicated in Table 1. All the DNP measurements were performed at the frequency maximizing positive polarization in NaAc at 3.46 T. The polarization build up was measured at 1.6-1.7 K resorting to small angle acquisition schemes of the .sup.1H NMR signal. By way of example, in
TABLE-US-00001 TABLE 1 Polarization level of .sup.1H (1.6-1.7K and 3.46 T) in the samples of Ex. 1-4 Days after Example P %* amorphization Storage 1 10 8 freezer 2 7.5 1 — 3 12 7 freezer 4 0.83 3 freezer *to be compared with thermal equilibrium P (0.21%).
Example 9
Physical Stability of Milled Lactose
[0154] After extraction from the cryogenic bath a first aliquot of the Lactose sample of Example 8 (Lactose-sample 1) was stored in a glove box (argon atmosphere, O.sub.2 and H.sub.2O content<0.1 ppm). An equivalent aliquot of the same sample (Lactose-sample 2) was left in the same room but outside the glove box. DNP measurements on the two samples of Lactose were then repeated after three weeks and after two months of ageing. The summary of the results obtained at 1.6-1.7 K is reported in table 2.
TABLE-US-00002 TABLE 2 Polarization level of .sup.1H (1.6-1.7K and 3.46 T) in DNP lactose samples upon ageing Days after Samples at 1.7K P %* amorphization Storage Lactose - sample 1 7.5 1 20° C. - inside GB Lactose - sample 1 7.5 20 20° C. - inside GB Lactose - sample 2 8.7 21 20° C. - outside GB Lactose - sample 1 8.9 72 20° C. - inside GB Lactose - sample 2 0.8 73 20° C. - outside GB GB = Glove Box; *to be compared with thermal equilibrium (0.21%).
[0155] As illustrated by the above table the polarization level of achievable on the aged samples inside or outside the glove box remains substantially constant after three weeks from the milling of the crystalline substrate, while after 2 months the sample kept out of the glove box shows a decrease of the polarization level.
Example 10
Rapid Dissolution of a Lactose Sample to Achieve a Ready-to-Inject Solution
[0156] 85 mg (about 0.25 mmol) of Lactose-TEMPO solid solution, prepared by co-milling as described in Example 2, is flashed frozen in a He-bath and polarized under standard DNP conditions (magnetic field 3.46 T, MW frequency 96.93 GHz, temperature 1.75 K) for 1 hour to allow both .sup.1H and .sup.13C nuclei to get to a hyperpolarized steady state, corresponding for the former nuclei to a polarization level of about 8%. At the end of the DNP procedure, the MW irradiation is switched off, the sample is raised 10 cm above the center of the magnetic field and dissolved by 5 ml of overheated water (about 10 bar, 170° C.) to obtain a liquid solution with 50 mM Lactose concentration, suitable for use in animal experiments (0.4 mmol/kg of Lactose for administered doses of 8 ml/kg).
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