Patent Application
20230400538
Magnetic resonance (MR) applications, including imaging and spectroscopic applications, have proven useful in diagnosis of many diseases. MR applications provide detailed images of soft tissues, e.g., abnormal tissues such as tumors and other structures, and characterization of the chemical composition of tissues such as small molecules involved in metabolic processes that may be altered in a diseased state, which cannot be readily assessed by other modalities, such as computed tomography (CT). MR is advantageous also because MR applications operate without exposing patients to ionizing radiation, compared to CT and positron emission tomography (PET).
Known compositions and preparation methods of MR probes are disadvantaged in some aspects and improvements are desired.
In one aspect, a composition is provided. The composition includes a magnetic resonance (MR) probe and a glassification agent. The glassification agent includes lactic acid.
In another aspect, a method of preparing a hyperpolarized magnetic resonance (MR) probe material is provided. The method includes preparing a composition including an MR probe, an electron paramagnetic agent (EPA), and a glassification agent. The glassification agent includes lactic acid. The method further includes obtaining a hyperpolarized amorphous solid MR probe material by carrying out polarization on the composition.
In yet another aspect, a method of preparing a hyperpolarized magnetic resonance (MR) probe is provided. The method includes preparing a composition including an MR probe, an electron paramagnetic agent (EPA), and a glassification agent. The glassification agent includes lactic acid. The method further includes carrying out polarization on the composition to obtain a hyperpolarized amorphous solid MR probe material. The method also includes liquefying the hyperpolarized amorphous solid MR probe material by dissolving the hyperpolarized amorphous solid MR probe material to obtain a hyperpolarized liquid MR probe solution.
Non-limiting and non-exhaustive embodiments are described with reference to the following Figures, wherein like reference numerals refer to like parts throughout the various drawings unless otherwise specified.
The present disclosure includes hyperpolarized MR probe compositions and methods for preparing hyperpolarized MR probe compositions. The compositions include lactic acid as a glassification agent. Method aspects will be in part apparent and in part explicitly discussed in the following description.
In MR applications, a subject is placed in a magnet. As used herein, a subject is a human, an animal, or a phantom, or part of a human, an animal, or a phantom. When the subject is in the magnetic field generated by the magnet, magnetic moments of nuclei, such as protons, attempt to align with the magnetic field but precess about the magnetic field in a random order at the nuclei's Larmor frequency. The magnetic field of the magnet is referred to as B0 and extends in the longitudinal or z direction. In acquiring an MRI image, a magnetic field (referred to as an excitation field B1), which is in the x-y plane and near the Larmor frequency, is generated by a radio-frequency (RF) coil and may be used to rotate, or “tip,” the net magnetic moment Mz of the nuclei from the z direction to the transverse or x-y plane. A signal, which is referred to as an MR signal, is emitted by the nuclei, after the excitation signal B1 is terminated. To use the MR signals to generate an image of a subject, magnetic field gradient pulses (Gx, Gy, and Gz) are used. The gradient pulses are used to scan through the k-space, the space of spatial frequencies or inverse of distances. A Fourier relationship exists between the acquired MR signals and an image of the subject, and therefore an image of the subject is derived by reconstructing the MR signals. In MR spectroscopy, scanning through the k-space with imaging gradients is not performed and the MR signals of selected voxels are used to generate spectra.
Typically, MR imaging (MRI) and spectroscopy (MRS) detect MR signals emitted from nuclei of hydrogen protons (1H) because the abundance of water in a subject. Carbon MR imaging or spectroscopy is of significant interest because carbon is the backbone of organic molecules and enables analysis of relevant biochemical pathways for disease monitoring and treatment in the human body as well as in non-human animal subjects. Carbon 12 (12C), however, is not detectable by MR due to the lack of spin in 12C. Although carbon 13 (13C) is detectable by MR, several factors limit 13C MRI/MRS. The gyromagnetic ratio of 13C is only approximately ¼ of 1H, greatly reducing magnitude of MR signals. Naturally occurring 13C is only 1.1% of carbon. Further, the centration of carbon in a subject is much lower than 1H. For example, a human body has 60-70% of water, in which the concentration of 1H is approximately 110 M, while the most concentrated metabolites are present in the mM ranges. As a result, MRI/MRS of endogenous 13C is impossible at reasonable imaging times.
Hyperpolarization of 13C MR probes significantly increases detectable 13C MR signals, for example, on the order of 104, thereby allowing in vivo 13C MRI/MRS within reasonable time. Hyperpolarized 13C MR probes are not radioactive. As a result, hyperpolarized 13C MR probes are safe to be used in vivo, advantageous over contrast agents used in positron emission tomography (PET) for studying metabolites.
Hyperpolarized 13C MR probes are also advantageous over other contrast agents for MR applications. Paramagnetic metal chelate complexes such as gadolinium chelates or superparamagnetic iron oxide particles are typical contrast agents used in MR applications. Typical MR contrast agents lack specificity, compared to hyperpolarized 13C MR probes. Further, toxic metal ions in paramagnetic metal chelate complexes may be released in the body after administration.
In the exemplary embodiment, the MR probe is a carbon and/or nitrogen-containing, biologically relevant, organic molecule that will enable metabolic characterization of a metabolic process. As used herein, a biologically relevant MR probe is a compound that is relevant to biological organs or tissues, such as humans, animals, plants and/or environmental health, and may be used to detect and/or indicate characteristics and functions in the biological organs or tissues. A biologically relevant MR probe may be an endogenous, naturally-occurring compound in the body, or a non-endogenous compound.
T1 is the time constant in the process by which the net magnetization returns to the initial value Mz, such that the MR signal of a hyperpolarized MR probe decays due to relaxation time T1 upon administration to a subject. Accordingly, the T1 value of an MR probe, in biological fluids such as blood, should be long enough to enable the probe molecule to be distributed to a target site in the subject and scanned while in the highly hyperpolarized state. Biologically relevant MR probes with suitable T1 relaxation times include carbonyl compounds because carbon atoms that do not have directly-attached protons, such as many carbonyl group carbons, generally have relatively long T1 relaxation times in the order of tens of seconds. For example, pyruvate has a T1 of 65 seconds at 3T. Biologically relevant non-carbonyl compounds having suitable T1 relaxation times are also contemplated herein as MR probes, such as cyclic carbohydrates and quaternary ammonium compounds.
In the exemplary embodiment, the MR probe is a carbonyl compound. The carbonyl compound may be selected from a carboxylic acid or urea. The carboxylic acid may be selected from maleic acid, acetic acid, fumaric acid, pyruvic acid, malonic acid. Succinic acid, oxaloacetic acid, lactic acid, ketoglutaric acid, nicotinic acid, alanine, glycine, cysteine, proline, tyrosine, sarcosine, gamma-aminobutyric acid (GABA), and homocysteine. Alternative carbonyl compounds with carbonyl carbons having either one or no directly-attached protons are also contemplated herein, for example aldehydes, nitrogenous carbonyls, ketones, amides, imines, and esters.
In the exemplary embodiment, hyperpolarization is achieved by dissolution dynamic nuclear polarization (DNP), which involves making a solution with highly polarized 13C and/or 15N nuclear spins on a carbon and/or nitrogen-containing molecule. An initial solution (unpolarized sample 16 in
In the exemplary embodiment, the EPA is a trityl radical. Oxygen-based, sulphur-based or carbon-based trityl radicals as EPA for hyperpolarization of MR probes via DNP results in high polarization levels for MR probes. In the exemplary embodiment, the EPA includes at least one of an oxygen-based trityl radical, a sulphur-based trityl radical, or a carbon-based trityl radical. Alternative EPA compounds are also contemplated herein.
Within the hyperpolarizer 14, the initial solution is cooled to approximately 1K within a magnetic field typically of 3.0-7.0 T, and solidified. The solid is subsequently irradiated with microwaves to transfer the high polarization of the free electrons in EPA to the MR probe's 13C and/or 15N nuclei, thus imparting the enhanced polarization to the MR probe.
In order for the DNP process to be effective, EPA should be in close proximity with MR probes to be polarized. The interaction between EPA and 13C MR probes is greatly increased in an amorphous solid compared to a crystalized solid. To facilitate the transfer of high levels of polarization in EPA to 13C MR probes, an amorphous solid should be formed in the hyperpolarizer 14. The process of forming an amorphous solid may be referred to as glassification. A glassification agent is often needed in a glassification process for the mixture of EPA and 13C MR probes to form an amorphous solid, instead of a crystalized solid. Water is a poor glassification agent because water crystallizes in a solid state.
Dissolution 22 of the now-hyperpolarized amorphous solid MR probe material (hyperpolarized solid 18 in
MR applications using hyperpolarized 13C MR probes are particularly powerful molecular characterization tools because MR using hyperpolarized 13C MR probes permits a safe, real-time or near real-time, non-radioactive, and pathway specific analysis of physiological processes that were previously inaccessible for spectroscopic and imaging applications. Similarly, the occurrence of nitrogen in biological systems makes nitrogen another species of interest for metabolic investigation using Nitrogen 15 (15N) nuclei MR probes, particularly in N-enriched probes subjected to hyperpolarization. There are, however, several technical challenges of known hyperpolarized MR probe compositions and methods.
In known MR probe compositions and methods, hyperpolarization of 13C and/or 15N NMR probes is adversely affected by the addition of water used to modify glassification agent concentration. For example, pyruvic acid has been demonstrated as a glassification agent during PA-urea co-polarization. Upon the addition of water to pyruvic acid, increased crystallization of the MR probe mixture occurred, resulting in decreased hyperpolarization of the MR probe. Further, in known MR probe compositions and methods, high concentrations of glassification agents, such as glycerol or pyruvic acid, needed for hyperpolarization may be toxic to a subject and/or cause metabolic perturbations in the processes being observed. For example, pyruvic acid concentrations in known MR probe compositions are multiple times higher than endogenous concentrations of pyruvic acid or conjugate base pyruvate in the human body. As another example, while glycerol is non-endogenous to the human body, glycerol is rapidly metabolized in the body and perturbs the metabolic processes being studied by MR. Also, glycerol has a relatively high viscosity and is difficult to reconstitute at dissolution. Additionally, in known MR probe compositions and methods, non-acidic glassification agents such as glycerol are unable to precipitate EPA or enable EPA removal via filtration from the hyperpolarized MR probe solutions. Following dissolution, EPA should be removed, such as to below 5 μM EPA or to below 3 μM EPA, before being injected into a subject. Moreover, in known MR probe compositions and methods, glassification agents are reactive with MR probes to form undesired reaction products in the MR probe mixtures. In addition, known MR probe compositions and methods include glassification agents with impurities that adversely affect glassification of MR probe mixtures. For example, in previous compositions including pyruvic acid as glassification agent with a urea 13C MR probe, three impurity compounds were identified as the cross-reaction product of pyruvic acid and urea.
In contrast, in the compositions and methods described herein, LA was surprisingly identified as a co-polarization probe and glassification matrix for 13C-15N MR probes, and was found to be more suitable than other glassification agents, such as pyruvic acid and glycerol, in several respects. First, adding water to MR probe compositions with LA as glassification agent does not cause crystallization and does not adversely affect the hyperpolarization of the MR probe. Further, glass formed using LA yield MR probes having desirable levels of polarization. Also, LA or conjugate base of lactate is endogenous to the human body at higher concentrations than endogenous pyruvic acid concentrations. In the exemplary embodiment, LA concentrations in MR probe compositions are comparable to endogenous LA concentrations, making MR probe compositions with LA safe and non-toxic. For example, a 0.06 mmol/kg dose of lactate did not result in metabolic perturbations as seen with pyruvic acid or pyruvate. Additionally, LA aids in EPA removal as the acidic component needed for filtration of EPA particles from hyperpolarized MR probe mixture dissolution product. EPA removal in the presence of LA is shown to be comparable to EPA removal in the presence of PA. LA-mediated EPA removal achieved acceptable levels below about 5 μM EPA. Moreover, LA is less reactive than PA, and LA contributes less impurities to MR probe compositions than PA. Urea/LA mixtures contain fewer impurities resulting from cross-reaction, and LA impurities do not adversely affect glassification of MR probe mixtures.
No known publications exist on using LA as a glassification agent. Surprisingly, LA MR probe compositions may be diluted with water without causing crystallization during hyperpolarization, and LA is advantageous over known glassification agents. The small amounts of impurities seen in LA were not adverse to glassification when LA was used as a glassification agent.
Accordingly, compositions and methods described herein overcome the above described problems in known MR probe compositions and methods by utilizing LA as a glassification agent. In the exemplary embodiment, LA is used as a glassification agent and/or co-polarization (e.g., co-hyperpolarization) agent for MR probe compositions and methods. Further details and examples are described below.
Glassification with LA upon hyperpolarization of 13C and/or 15N MR probes. LA was utilized as a glassification agent for urea MR probe solutions. Based on differential scanning calorimetry (DSC) results described herein, LA/urea mixtures at ˜3.2:1 molar ratio formed a glass. By using LA, the final dissolution product of urea was increased by >30% to 45-67 mM. DSC profiles were run for solutions spanning molar volume ratios of 0.8:1 to 7.5:1 LA:urea. Tests were performed using [1-12C] LA and 12C urea. Urea in water was first prepared and fully dissolved before mixing with LA. Samples were loaded at room temperature, heated and held isothermally for 30 minutes prior to cooling to below freezing. Solutions were held isothermally for 5 minutes below freezing and then re-heated with a subsequent 30 minute isothermal hold to ensure complete sample melting. The process was repeated for each sample and a constant ramp rate of 5° C./minute was used. Results are shown in
Reduced metabolic perturbation using LA as glassification agent. While a typical glassification agent such as glycerol is useful for reducing ice crystal formation during freezing and storage, glycerol is rapidly metabolized in vivo, may be toxic to the subject, and causes perturbations to the metabolic measurements being studied, such as TCA cycle measurements. LA is an endogenous compound in the human body. Because LA concentrations in MR probe compositions (refer to Table 1) are comparable to LA concentrations in the body, LA does not perturb metabolic reactions being observed. In the exemplary embodiment, MR probe compositions include a glassification agent that includes LA at a concentration up to about 90% volume percent LA based on a total volume of the composition.
Polarization using LA as glassification agent. Polarization with LA was evaluated and small dose LA/urea buildup and dissolution with LA was assessed. [13C,15N] urea in L-[1-13C] LA mixtures or [1-12C] LA mixtures were polarized at <1 K. 4.2:1 and 3.2:1 LA/urea molar ratio mixtures were prepared by adding urea solution to crystalline LA, see Table 1 below. The LA/urea final mixtures has an estimated density of 1.23-1.3 g/ml depending on LA used and mixture ratio. EPA was added to a final concentration of about 15 mM or less EPA.
Samples were polarized with microwave irradiation at an optimal frequency for urea for several hours, then rapidly dissolved in super-heated, pressurized sterile water and subsequently neutralized using neutralization buffer containing NaOH and Tris buffer. Fluid path was used alone or together with an EPA filter for EPA removal.
The various mixing ratios used in the DSC study along with corresponding testing results are shown in Table 2 and
As a further example, a small dose experiment using a 4.2:1 [1-13C] LA/[13C,15N] urea mixture with EPA was used to investigate solid phase buildup in a hyperpolarizing system and a dissolution experiment without EPA filtration was further performed. The sample vial was elevated higher than the typical lowered position in order to center the sample within the NMR coil so that solid phase buildup from a small sample may be monitored with a good signal to noise ratio. The optimal microwave frequency for urea was used, which is 0.01 GHz lower than 13C-pyruvic acid. A time constant of 2905s was reported, see
Dissolution medium was loaded into the dissolution syringe. Dissolution after 4 hours of buildup went smoothly and the dissolved LA and urea mixture was received in a bottle containing neutralization medium. Liquid phase polarization (LSP) was estimated. LSP indicates the extent of polarization of nuclei (e.g., 13C or 15N nuclei) in an MR probe, and may be expressed as the percentage of total nuclear spins that have become highly polarized in an MR probe, such that a higher LSP results in a higher MR signal. In the exemplary embodiment for injection into a subject, a hyperpolarized 13C MR probe should have an LSP of at least about 20%, where at least about 20% of the total 13C nuclei in the MR probe have highly polarized 13C nuclear spins. LSP was monitored with the first measurement started at 91 seconds. Additional samples were tested every minute after the first measurement to monitor signal decay. LSP was estimated by comparing the hyperpolarized product spectrum to the thermal-equilibrium spectrum and then back-calculating to the time of dissolution using T1 time constants fit by the measurements, see
Polarization and dissolution with LA as glassification agent.
EPA removal using LA as glassification agent. EPA removal performance with LA was evaluated. In MR probe compositions including pyruvic acid, EPA forms particles in the presence of pyruvic acid during dissolution which then are filtered out using pharmacy kit filters. The residual EPA concentration in the dissolution product is typically less than 3 μM. Clinical site acceptance criteria ranges from about ≤5 μM to about ≤3 μM for EPA concentration in the final injectable solution. During co-polarization of pyruvic acid-urea protocol, starting EPA concentration was below mM and final EPA concentrations below about 3 μM were reported.
While LA has a higher pKa value (3.8) than pyruvic acid (2.45), the acidic condition imparted by LA was shown to be suitable in forming EPA particles for removal via filtration. Previous work has shown that fumaric acid with pKa>3.0 (e.g., pKa=3.03, 4.44) resulted in residual EPA at a few μMs higher than the typical concentration from pyruvic acid dissolution. In dissolution runs described herein with 4.2:1 and 3.2:1 LA/urea ratio, 12C LA was used instead of the high cost 13C version.
Table 4 shows formula and dissolution results with [13C,15N] urea prepared in 12C LA. A concentration of 12 mM EPA was used in the starting mixture, similar to concentrations typically used for the study of pyruvic acid/urea mixtures. For 12 mM EPA concentration, the solid phase polarization built up slower but reach a higher level. The residual EPA concentration from 3.2:1 ratio mixture is higher than 4.2:1 mixture since less LA is present. Therefore, according to the acceptance criteria for human subject injection of ≤5 μM EPA, the LA formulations of the present disclosure are acceptable. The 3.2:1 ratio provided 141 mM lactate and 42 mM urea at 80% recovery. Exemplary embodiments of the present disclosure utilizing LA co-polarization may provide 20-40% higher concentration of urea relative to the above-described example of 35 mM [13C,15N] urea from pyruvic acid co-polarization.
12C-lactate
13C,15N-urea
In the exemplary embodiment, the use of LA either as a 12C glassification agent in a [13C—15N] urea solution and/or as a 13C co-hyperpolarization probe warrants quality control checks to ensure non-harmful LA and EPA concentrations prior to subject injection. As another example, LA absorbance may be suitably monitored provided that a UV-transparent cuvette (e.g., quartz) is used. Maximum absorption by LA occurs in the ultra-violet range (<230 nm), which is below the transmission range for disposable methacrylic or polysterene cuvettes as in previously used compositions and methods to measure pyruvic acid.
Preliminary absorbance measurements of LA were carried out using 1-cm quartz cuvettes (non-disposable). Additional spectra were obtained using disposable cuvettes and results for both tests are shown in
LA-associated impurities and LA-associated side reaction products. NMR studies of 13C LA with or without urea in water were performed to evaluate the contribution of LA-associated impurities side reaction products with MR probes. Potential contaminants within the LA and potential reaction products (e.g., between LA+urea, LA+EPA, LA+neutralization buffer(s)), were identified. Identified contaminants and/or reaction products were examined via time-series. Starting materials included 13C LA and 13C-15N Urea, results spectra are shown in
13C-LA both without and with concentrated EPA.
13C-LA
13C-LA-concentrated-EPA
13C-Urea-13C-15N + LA mixture
13C-Urea-13C-15N + LA mixture-EPA
Accordingly and based on the above, one or more technical effects of the compositions and methods described herein include: (a) glassification with LA during hyperpolarization of 13C and/or 15N MR probes, (b) EPA removal via filtration in the presence of LA, (c) non-toxic MR probe mixtures with LA, (d) hyperpolarization of MR probe mixtures with LA not being adversely affected by addition of water, (e) improved MR analysis due to decreased metabolic perturbations with LA, (f) reduced impurity contributions by LA, and (g) reduced undesired reaction products in MR probe mixtures with LA.
Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
In the exemplary embodiment, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, that may be used to describe and claim aspects of the present disclosure are to be understood as being modified in some instances by the term “about.” The term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The numerical parameters set forth in the written description and attached claims are approximations that vary depending upon the desired properties sought to be obtained by a particular aspect of the disclosure. The numerical parameters are be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of aspects of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in aspects of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
The terms “a” and “an” and “the” and similar references used in the context of describing a particular aspect (especially in the context of certain of the following claims) may be construed to cover both the singular and the plural, unless specifically noted otherwise. The term “or” as used herein, including the claims, may be used to mean “and/or” unless explicitly indicated to refer to alternatives only or to refer to the alternatives that are mutually exclusive.
The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and may also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and may cover other unlisted features.
All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to exemplary embodiments described herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
Groupings of alternative elements or aspects of the present disclosure disclosed herein are not to be construed as limitations. Each group member is referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group are included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
To facilitate the understanding of the aspects described herein, a number of terms are defined below. The terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. Terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity, but rather include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific aspects of the disclosure, but terminology usage does not delimit the disclosure, except as outlined in the claims.
All of the compositions and/or methods disclosed and claimed herein may be made and/or executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of the aspects included herein, it will be apparent to those of ordinary skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the disclosure as defined by the appended claims.
This written description uses examples to disclose the disclosure, including the best mode, and also to enable any person skilled in the art to practice the disclosure, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the disclosure is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.
This invention was made with government support under grant number R01CA214554 awarded by the National Institutes of Health. The government has certain rights in the invention.
