This application claims the benefits of U.S. Provisional Patent Application No.

62/825,963 filed on March 29, 2019 which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods for detecting and imaging molecules that are present in a non living sample or a living organism, and in particular, detecting and imaging molecules, or compositions of molecules. Embodiments of the present invention employ compounds containing a ketone functional group and a radioactive or nonradioactive tracer moiety in conjunction with an imaging device such as a Positron Emission Tomography (PET) and/or a Magnetic Resonance Spectroscopic Imaging (MRS I).

BACKGROUND OF THE INVENTION

Clinical imaging is well known and widely used for diagnostic and therapeutic purposes. For example, altered levels of glucose metabolism are known to be associated with the presence of various cancers, seizures, dementia and other disease states. The in vivo detection and monitoring of glucose metabolism can be accomplished via the administration of a molecule with a radioactive tracer, such as fludeoxyglucose F ¹⁸ , also known as 2-deoxy-2- [ ¹⁸ F]fluorodeoxyglucose (hereinafter“F ¹⁸ DG”) to a patient and constructing a three dimensional image of the tracer concentration within the body employing a PET scanner. The image generated by the F ¹⁸ DG concentration allows the medical professional to determine the location of a cancerous tumor and the extent of metastasis. The use of nonradioactive tracer compounds such as fludeoxyglucose F ¹⁹ , also known as 2-deoxy-2-[ ¹⁹ F]fluorodeoxyglucose (hereinafter“FDG”) has also been reported in the literature. For example, U.S. Patent Nos. 9,207,298 and 9,014,785, which are incorporated herein by reference, describe the use of FDG as a tracer for use in MRSI imaging techniques.

U.S. Patent No. 8,642,654, which is incorporated herein by reference, describes ketone bodies that may be used as a source of energy in most tissues of the body and suggests that the administration of pharmaceutically and physiologically safe ketone bodies can lead to various clinical benefits, including an enhancement of physical and cognitive performance and the treatment of cardiovascular conditions, diabetes, neurodegenerative diseases and epilepsy.

SUMMARY OF THE INVENTION

The present disclosure relates to the use of ketone bodies as a tracer compound for PET and MSRI imagining. The ketone bodies are modified to include a radioactive or nonradioactive tracer moiety.

In certain embodiments, the ketone bodies contain one or more F ¹⁸ or F ¹⁹ atoms.

In certain embodiments, the ketone bodies contain one or more F ¹⁸ atoms and may be administered, orally or parenterally, to a patient or subject in need of imaging using PET techniques.

In certain embodiments, the ketone bodies contain one or more F ¹⁹ atoms and may be administered, orally or parenterally, to a patient or subject in need of imaging using MSRI techniques.

In certain embodiments, the ketone bodies are labelled with an isotope of either radioactive C ¹⁴ , or nonreactive C ¹³ and may be administered orally or parenterally, to a patient. C ¹⁴ will normally be measured by scintillation, whereas C ¹³ is detected by isotope mass spectrometry.

DETAILED DESCRIPTION OF THE INVENTION

Except where noted, all terms are intended to have their normal meaning in the art, and are used as they would have been used by a person of ordinary skill at the time of the disclosure. It should be understood that throughout this application the singular forms, such as“a,”“an,” and “the,” are often used for convenience, however, these singular forms are intended to encompass the plural unless otherwise specified, or unless the context clearly calls for the singular alone. It should also be understood that all publication, patents, books, journal articles, and the like, which are referred to in this application, are incorporated by reference in their entirety and for all purposes to the extent not inconsistent with the present disclosure.

Concentration, weight percent and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a percent range of 1% to 20% should be interpreted to include not only the explicitly recited percent limits of 1% and 20% but also to include individual percentages such as 1.25%, 2.49%, 3%, 4.75%, 8.34% and sub-ranges such as 1% to 5%, 10% to 15%, 4.7% to 11.9% etc.

The terms“individual,”“subject,” or“patient” are used interchangeably. As used herein, they mean any mammal including but not limited to humans, dogs, cats, horses, cows, monkeys, rabbits, mice, and rats and guinea pigs. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human.

“Effective amount” means the amount of a ketone body that has been modified to contain a tracer moiety and administered to a subject which will allow the medical provider or technician to obtain images of a subject’s body, body region, or body part using a specific imaging technique such as PET or MSRI. The effective amount will vary depending on the tracer moiety employed in the ketone body molecule, the subject’s individual characteristics such as sex, age, weight, the desired area for imaging and the specific imaging technique.

“Administering” and related terms such as “administration,” “administer” and “administered” includes any mode of administration or methods for introducing an effective amount of the ketone bodies into a subject’s body and preferably a subject’s circulatory system. The methods for administering include known methods such as oral, parenteral, vaginal, rectal, ophthalmic, otic, nasal, and inhalation. Parenteral methods of administration include introducing the desired amount of the ketone bodies to a subject via an injection such as subcutaneous, intramuscular, intraorbital, intracap sular, intraspinal, intrastemal or intravenous injection. Parenteral administration also includes adding, mixing or diluting a composition containing the desired amount of the ketone bodies into an intravenous solution such as a Ringer’s solution or 0.9% w/v saline solution.

As used herein the term“ketone body” or“ketone bodies” refers to any molecule that contains a ketone moiety, i.e., a carbonyl group bonded to two carbon atoms (R ₂ -C=0 wherein R is not H) and that may be used as a source of energy in most animal tissues. Examples of ketone bodies are provided in U.S. Patent No. 8,642,654, U.S. Patent Application Publication Nos. 2017/0196827 and 2012/0064611 and International Patent Application No. WO 2017/119986 which are incorporated herein by reference. Specific examples of ketone bodies that may be used in the present invention include but are not limited to (R)-3-hydroxybutyrate, acetoacetate, 3- hydroxybutyl 3-hydroxybutyrate and the isomers thereof including (3R)-hydroxybutyl(3R)- hydroxybutyrate. A particularly preferred ketone body is a compound of formula I:

In accordance with the present invention, the ketone body compound will be modified to incorporate a tracer moiety which may include a radioactive isotope such as C ¹¹ , C ¹³ , C ¹⁴ , N ¹³ , O ¹⁵ , F ¹⁸ , I ¹²⁴ or a combination thereof. Alternatively, the ketone body will include a nonradioactive tracer moiety such a F ¹⁹ .

In a preferred embodiment, the ketone body will be selected form the group consisting of 3-hydroxybutyrate, acetoacetate, 3-hydroxybutyl 3-hydroxybutyrate, isomers of the forgoing and combinations thereof wherein one or more of the hydrogen atoms or hydroxyl groups is replaced with F ¹⁸ . In a particularly preferred embodiment, the ketone body is a compound of formula I wherein one or more of the hydrogen atoms or hydroxyl groups is replaced with F ¹⁸ .

In another embodiment, the ketone body will be selected form the group consisting of 3- hydroxybutyrate, acetoacetate, 3-hydroxybutyl 3-hydroxybutyrate, isomers of the forgoing and combinations thereof wherein one or more of the hydrogen atoms or hydroxyl groups is replaced with F ¹⁹ . In a particularly preferred embodiment, the ketone body is a compound of formula I wherein one or more of the hydrogen atoms or hydroxyl groups is replaced with F ¹⁹ . In yet another embodiment, the ketone body will be selected form the group consisting of 3-hydroxybutyrate, acetoacetate, 3-hydroxybutyl 3-hydroxybutyrate, isomers of the forgoing and combinations thereof wherein one or more of the carbon atoms are replaced with C ¹³ or C ¹⁴ .

The ketone body with a tracer moiety may be prepared by any method known in the art. For example methods for preparing the ketone body of formula I are provided in U.S. Patent Application Publication No. 2012/0064611. Once the compound of formula I is prepared, one of the hydroxyl groups may be protected with a protecting group such as an acetyl protecting group and the other hydroxyl group may be replaced with a triflate. The hydroxyl protected, triflate derivative of the compound of formula I is then reacted an F ¹⁸ source such as [(crypt- 222)K] ^{+ 18} F so the F ¹⁸ anion replaces the triflate moiety and the cryptand and hydroxyl protecting group are removed to produce the F ¹⁸ ketone body of a compound of formula I that may be used in the methods of the present invention. A skilled artisan would also know how to incorporate one or more F ¹⁹ atoms into a ketone body, including a compound of formula I using the methods described herein for producing F ¹⁸ ketone bodies.

The ketone bodies with the tracer moiety, preferably an F ¹⁸ or F ¹⁹ tracer moiety, may be administered to a subject either orally or parenterally. The oral administration may comprise the step of orally administering to a subject a solid or liquid composition comprising an effective amount of the ketone body with the tracer moiety and at least one pharmaceutically acceptable excipient such as a carrier, diluent, binder, glidant, solubilizing agent, wetting agent, emulsifier, preservative, lubricant, flavoring agent, pH adjusting agent, or combinations thereof. Similarly the parenteral administration may involve the step of parenterally administering, preferably intravenously, a composition comprising an effective amount of the ketone body with the tracer moiety and at least one pharmaceutically acceptable excipient such as a carrier, diluent, buffer, solubilizing agent, wetting agent, emulsifier, preservative, tonicity agent (osmolality agent), pH adjusting agent, or combinations thereof. Examples of the various excipients that may be employed in the compositions described herein can be found various reference materials such as the United States Pharmacopeia (“USP”) 29 (2006) pp. 3256-3261 and Rowe et ah, eds., Handbook of Pharmaceutical Excipients, 6 ^th ed. 2009.

In certain embodiments, the parenteral compositions comprise: (i) an effective amount of the ketone body with the tracer moiety; (ii) a liquid carrier such as water, glycerin, polyethylene glycol or combinations thereof; and (iii) optionally one or more pharmaceutical excipients selected from the group consisting of a buffer, a solubilizing agent, a wetting agent, an emulsifier, a preservative, a tonicity agent (osmolality agent), a pH adjusting agent, or combinations thereof. As indicated above the parenteral composition may also comprise an effective amount of the ketone body dispersed or dissolved in a conventional intravenous fluid such as 0.9% w/v saline or Ringer’s solution.

In certain embodiments, the oral compositions comprise: (i) an effective amount of the ketone body with the tracer moiety; (ii) a solid or liquid carrier and (iii) optionally one or more pharmaceutical excipients selected from the group consisting of a buffer, a solubilizing agent, a wetting agent, an emulsifier, a preservative, a tonicity agent (osmolality agent), a pH adjusting agent, a thickening or viscosity enhancing agent or combinations thereof. If the oral composition is a liquid composition, it may be a solution, suspension or emulsion. Examples of liquid carriers that may be used in the liquid oral formulations, include but are not limited to water and polyols such as, glycerin, polyethylene glycol or combinations thereof. If the oral composition is a solid composition, it may be a tablet, capsule, powder, pellets or granules. Examples of solid carries that may be used in the solid oral formulations include celluloses such as powdered cellulose, microcrystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl ethylcellulose, and methylcellulose, croscarmellose sodium, crospovidone, starch, pergelatinized starch, sodium starch glycolate, calcium carbonate, calcium phosphate, calcium sulfate, kaolin, and combinations thereof. The solid oral compositions may further optionally, comprise a binder, diluent, lubricant or combination thereof. The solid oral compositions should dissolve or disintegrate within 60 minutes or less, preferably 45 minutes or less and more preferably within 30 minutes of less when tested with an appropriate United States Pharmacopeia dissolution or disintegration testing apparatus.

In certain embodiments, it is desired that the compositions comprising the effective amount of the ketone bodies with a tracer moiety be free of any sugar such as glucose, sucrose, lactose, etc. which would interfere with the absorption and/or action of the ketone body at the desired location such as a tumor.

In the case of ketone bodies with a radioactive tracer moiety such as F ¹⁸ , the effective amount of the ketone body with an F ¹⁸ tracer moiety that may be administered either orally or parenterally, preferably intravenously, should range from an amount about 150 to about 600 MBq of F ¹⁸ , preferably about 200 to about 500 MBq of F ¹⁸ and more preferably about 250 to about 450 MBq of F ¹⁸ . If the ketone bodies with an F ¹⁸ tracer moiety is employed in the methods of the present disclosure, the administration should occur when the subject is in a fasted stated. More specifically, the subject should have refrained from eating or drinking anything except water or black coffee for at least 4-12 hours prior to administration of the ketone body and preferably at least 4-6 hours prior to admiration of the ketone body. It is also desirable that subject’s blood glucose level be less than 160 mg/dL, preferably less than 150 mg/dL and most preferably less than 140 mg/dL at the time of administration of the ketone body. After the effective amount of the ketone body with the tracer moiety is administered, the imaging using a device such as a PET scanner should begin within 20-120 minutes after administration, preferably, within 30-90 minutes after administration and most preferably within 30 to 60 minutes after administration.

In certain embodiments, about 250 to about 450 MBq of one or more ketone bodies with an F ¹⁸ tracer moiety are administered to human subjects in a fasted stated and after about 30 to 90 minutes of administration the subject receives a PET or PET/CT scan and the resulting images may be used to:

i. Differentiate or diagnosis benign from malignant lesions or tumors;

ii. Locate an unknown primary tumor when metastatic disease is discovered as the first manifestation of cancer or when the subject presents with a paraneoplastic syndrome;

iii. Monitor or measure the effect of therapies on known malignancies;

iv. Determine optimal sites for a biopsy;

v. Guide radiation therapy;

vi. Evaluate or diagnosis infections

vii. Evaluate or diagnosis cardiovascular conditions such as atherosclerosis;

viii. Evaluate or diagnosis neurodegenerative conditions; and

ix. Evaluate or diagnosis seizures and epilepsy.

In the case of ketone bodies with a radioactive tracer moiety such as F ¹⁹ , the effective amount of the ketone body with an F ¹⁹ tracer moiety that may be administered either orally or parenterally, should range from an amount about 100 mg to about 2,500 mg, preferably about 250 mg to about 2,000 mg and most preferably about 500 mg to about 1,500 mg. If the administration is oral the total dose of the ketone body with an F ¹⁹ tracer moiety may be administered in a single or divided doses. For example a total of 2,000 mg may be orally administered by having the patient swallow four (4) tablets containing 500 mg of the ketone body with an F ¹⁹ tracer moiety. If the ketone bodies with an F ¹⁹ tracer moiety is employed in the methods of the present disclosure, the administration should occur when the subject is in a fasted stated. More specifically, the subject should have refrained from eating or drinking anything except water or black coffee for at least 4- 12 hours prior to administration of the ketone body and preferably at least 4-6 hours prior to admiration of the ketone body. It is also desirable that subject’s blood glucose level be less than 160 mg/dL, preferably less than 150 mg/dL and most preferably less than 140 mg/dL at the time of administration of the ketone body. After the effective amount of the ketone body with the tracer moiety is administered, the imaging using a device such as an MSRI scanner should begin within 20-120 minutes after administration, preferably, within 30-90 minutes after administration and most preferably within 30 to 60 minutes after administration. The ketone body with an F ¹⁹ tracer moiety may be used to generate images to:

i. Differentiate or diagnosis benign from malignant lesions or tumors;

ii. Classify solid tumors as either glucose positive or ketone positive;

iii. Locate an unknown primary tumor when metastatic disease is discovered as the first manifestation of cancer or when the subject presents with a paraneoplastic syndrome;

iv. Monitor or measure the effect of therapies on known malignancies;

v. Determine optimal sites for a biopsy;

vi. Guide radiation therapy;

vii. Evaluate or diagnosis infections

viii. Evaluate or diagnosis cardiovascular conditions such as atherosclerosis; ix. Evaluate or diagnosis neurodegenerative conditions; and

x. Evaluate or diagnosis seizures and epilepsy.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following are provided by way of example only and are by no means intended to be limiting.

Example 1

An F ¹⁸ ketone body may be prepared by the general reaction scheme shown below:

wherein Ac is CH ₃ CO and Tf is CF ₃ SO ₂ .

Example 2

An F ¹⁸ ketone body may be prepared by the general reaction scheme shown below:

wherein Ac is CH ₃ CO and Tf is CF ₃ SO ₂ .

Example 3

An F ¹⁸ ketone body may be prepared by the general reaction scheme shown below:

wherein Ac is CH ₃ CO and Tf is CF ₃ SO ₂ . Example 4

The ketone bodies with F ¹⁸ tracer moieties prepared in Examples 1, 2 or 3 may be mixed with 0.25 to 1 ml of USP water for injection and about 300 to 400 MBq of the F ¹⁸ ketone bodies are intravenously administered to an adult male or female subject that fasted for 4-6 hours prior to administration. After 30 to 60 minutes the subject may receive a full body PET scan.

Example 5

The ketone bodies with F ¹⁹ tracer moieties prepared according to the above procedures of Examples 1, 2 or 3 wherein the F ¹⁸ is replaced with F ¹⁹ .

Example 6

Tablets containing the ketone bodies with F ¹⁹ tracer moieties prepared in Example 4 can be prepared using conventional tableting techniques such as wet and dry granulation and/or slugging and compressing the results granules or powders into tablets using a conventional tablet press. The tablets may have the following compositions:

Example 7

The ketone bodies with F ¹⁹ tracer moieties prepared in Example 5 may be mixed with conventional excipients as previously described to prepare capsules, powders, solutions, suspensions or tablets as described in Example 6 orally administered in an amount of about 1,000- 1,500 mg to an adult male or female subject that fasted for 4-6 hours prior to administration. After 30 to 60 minutes the subject may receive an MSRI scan.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms“comprising,”“consisting essentially of’ and“consisting of’ may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.