Carbon 13-labelled taggant for determining adherence to or unauthorised use of a pharmaceutical dosage regimen

Abstract

The present invention relates to a pharmaceutical composition comprising a biologically active agent and one or more carbon 13 labelled taggant as defined herein. The present invention further relates to a method of determining adherence to, or unauthorised use of, a pharmaceutical dosage regimen comprising use of said pharmaceutical composition, a system for identifying use of a biologically active agent, and the use of a taggant as defined herein for preparation of said pharmaceutical composition. In one aspect, the taggant is a compound of formula (1): or a pharmaceutically acceptable salt thereof, and wherein the compound of formula (1) contains at least one carbon 13 atom.

Description

[0001] TAGGANT

[0002] The present invention relates to a pharmaceutical composition comprising a biologically active agent and one or more carbon 13 labelled taggant as defined herein. The present invention further relates to a method of determining adherence to, or unauthorised use of, a pharmaceutical dosage regimen comprising use of said pharmaceutical composition, a system for identifying use of a biologically active agent, and the use of a taggant as defined herein for preparation of said pharmaceutical composition.

[0003] BACKGROUND OF THE INVENTION

[0004] In 1985 Surgeon General Everett Koop stated that, “Drugs don’t work in patients who don’t take them.” Patients may have countless accidental or intentional reasons for not taking a medication as prescribed. Even under direct observation it is difficult to conclusively determine that a patient consumed a prescribed pill or otherwise introduced a prescribed biologically active agent into their body (Zullig, L.L., Mendys, P. and Bosworth, H.B., 2017. Medication adherence: A practical measurement selection guide using case studies. Patient Education and Counseling, 100(7), pp.1410-1414.). Intentional reasons for not using medications include, but not limited to, undesirable side effects of a medication, stockpiling a drug to misuse it at another time, or opportunities to sell or trade a drug to someone else. Some medical personnel may even take opportunities to substitute a prescribed drug with counterfeit drug or placebo thereby allowing them to divert the actual chemical from the intended patient. This invention describes a method to combine fast acting stable isotope taggants with non-invasive breath testing to allow verification that a prescribed therapeutic was successfully introduced into the body of a patient.

[0005] Previous patent applications have documented the large-scale health and economic impacts of failed adherence to prescribed drug regimens (US200 / 70224128 A1 ; WO2015 / 134390 A1 ; US9047746B1). To summarize, non-compliance has resulted in an estimated $100 billion in excessive healthcare costs per year and has caused approximately 125,000 deaths in the U.S. annually. Furthermore, 25% of healthcare costs could be avoided with proper compliance. The safety issues of adherence and compliance are particularly important for drug trials with paid participants where the motivation to adhere to a specific drug dosing regimen may not be correlated with actual medical need. As a result, several technologies have been created to address issues of drug compliance and adherence (Vik, S.A., Maxwell, C.J. and Hogan, D.B., 2004. Measurement, correlates, and health outcomes of medication adherence among seniors. Annals of Pharmacotherapy, 38(2), pp.303-312.).

[0006] One group of solutions to monitor oral drug compliance involves a capsule that is wrapped in an electronic microcircuit which is activated upon exposure to gastric fluids and subsequently detected by a receiving device outside of the body which relays information to a medical provider (US 9047746 B1). Newer versions of ‘electronic pills’ expand on these features by enabling messaging and reminders on the users’ mobile communication devices (US2013 / 0117696 A1). Unfortunately, likely due to safety concerns and cost, these solutions have not been widely adopted. Moreover, these ingestible sensors are only suited to monitor oral medications delivered in solid form (Eliasson, L., Clifford, S., Mulick, A., Jackson, C. and Vrijens, B., 2020. How the EMERGE guideline on medication adherence can improve the quality of clinical trials. British Journal of Clinical Pharmacology, 86(4), pp.687-697.).

[0007] There are several other examples of inventions that enable chemical monitoring body fluids (blood, breath, saliva, etc.) to monitor adherence to drug therapies. Breath testing methods for monitoring drug compliance may measure exotic volatile organic compounds or non-ordinary stable isotopes of hydrogen, carbon, or oxygen present in either the CO2or the H2O of the breath.

[0008] The non-ordinary stable form of hydrogen is deuterium and is constitutively present in measurable amounts in exhaled water vapor in all humans. An existing invention takes advantage of the fact that a deuterated taggant can be dosed alongside a drug and the presence of deuterium in either exhaled organic molecules or water vapor can be used, in theory, to confirm delivery of the drug (US20180017569A1 ; JP2010519553A5).

[0009] Unfortunately, deuterium, like its isotopic sibling hydrogen, is a labile atom often loosely bound to a larger molecule and tends, even in the absence of enzymatic reactions, to be readily exchanged with the hydrogen atoms of nearby organic molecules as well as surrounding water molecules which comprise most of the body mass. As such, deuterium delivered as a drug taggant does not necessarily ‘wash out’ of the body in predictable ways leading to a gradual accumulation of deuterium in the molecules of the body, limiting its value as a useful taggant in medications intended to be taken daily. Similar ‘bioaccumulation’ of non-ordinary13C atoms can also occur in other taggant approaches (see specific examples below).13C-taggants are highly effective because carbon exchange, unlike hydrogen exchange, requires enzymatic processes. Another advantage of using13C in a taggant is that highly enriched13C-containing molecules cannot be found in nature and the primary method to obtain highly enriched13C is through industrial cryodistillation of13C-carbon monoxide. As such, sources of13C are well controlled and this drug delivery verification method would be difficult to counterfeit. Other patented methods use13C-lipid,13C-carbohydrate, or deprotein taggants that can be detected in the exhaled CO2. Unfortunately, likely due to the large amounts of taggant required, the high cost of the tracer molecules, and / or technological limitations with detecting the target molecules in trace amounts, these methods have not become widely adopted.

[0010] A seminal invention in the field of breath testing outlined the use of13C-labeled lipid taggants (including glycerides [monoglycerides / diglycerides / triglycerides], phospholipids, glycolipids, and fatty acids [preferably medium and long chain fatty acids]) to confirm ingestion of a medication to monitor compliance with medical prescriptions (US20060188444A1). The claim is that the13CO2released in the breath is the result of p- oxidation of carbon units from lipid chains. While the claim is technically correct, the time course of13C-lipid taggant ingestion, subsequent absorption, and ultimately p-oxidation within target tissues (typically the liver) is a slow process requiring several hours to generate detectable13CO2enrichment in the breath (see Figure 1). As described in US20060188444A1 , the timing of the peak13CO2values of13C-lipid taggants occurs only after 7-9 hours following ingestion. This time course is not suitable for reasonably rapid detection of ingested therapeutics.

[0011] The large bolus (“preferably 100-200 mg” [US20060188444A1]) of13C-labeled taggant required by the existing art to elicit the minimal response to confirm drug delivery is typically larger than the amount of active drug that it is designed to track. In contrast, the small-molecule taggants outlined in the present invention are more potent thereby requiring smaller boluses, and they are much faster acting than13C-lipid taggants.

[0012] Another issue with US20060188444A1 is that the13C-lipid tracers are not rapidly cleared from the body causing the13C-lipid taggants to gradually accumulate into lipid components of all body tissues. The implication is that the body tissues (notably the lipids stores) gradually become elevated in13C (McCue, M.D., Amaya, J.A., Yang, A.S., Erhardt, E.B., Wolf, B.O. and Hanson, D.T., 2013. Targeted13C enrichment of lipid and protein pools in the body reveals circadian changes in oxidative fuel mixture during prolonged fasting: a case study using Japanese quail. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 166(4), pp.546-554; Levin, E., McCue, M.D. and Davidowitz, G., 2017. More than just sugar: allocation of nectar amino acids and fatty acids in a Lepidopteran. Proceedings of the Royal Society B: Biological Sciences, 284(1848).) thereby precluding the effective use of the taggant on a prolonged basis. In contrast, the present invention differs in that it does not involve13C-lipid taggants which will gradually accumulate in the subjects’ tissues over long periods. Moreover, the13C- labeled-taggants of the present invention do not require p-oxidation, which is a slow / protracted biological process, to gradually enrich the breath. Conversely, meaningful13C -enrichments can typically be observed within a few minutes following ingestion of the taggants of the present invention.

[0013] Interestingly, US20060188444A1 also states that the use of alternative13C-taggants (specifically13C-carbohydrates or13C-amino acids) is unsuitable to monitor medical compliance because "...the metabolism rate is too rapid to cause continuous discharge of isotope-labelled CO2in the exhaled air, making it difficult to monitor patient or subject compliance with medical prescriptions." However, the empirical data presented in the present application demonstrates certain disadvantages of13C-lipid taggants as compared to targets of the present invention.

[0014] GB2442980A describes the invention of an ‘interlock’ device designed to fit into a vehicle that detects stable (i.e.,18O or13C) or radioactive isotopes (e.g.,14C) in the breath released from drugs known to impair driving ability. That invention involves mixing a rare isotope with the mind-altering drug (e.g., sedatives, pain killers, etc.), preferably in the form of glucose or other monosaccharides. The inventors advocate the use of these simple carbohydrates in place of more complex carbohydrate taggants to maximize the speed of13CO2generation and release in the breath. It is noteworthy that GB2442980A does not provide any empirical data of the time course or magnitude of13CO2generation, underscoring a limited understanding on the nuances of ‘cheaper, more ‘rapid’ and more ‘potent’13C-taggants described in the present invention. Moreover, the present invention differs from GB2442980A in that it does not involve the use of13C-carbohydrate taggants, but rather a class of taggant that will generate more rapid and stronger13C-signals in the breath at lower costs.

[0015] A nutritional research study in Mexico employed13C-taggants to monitor adherence to experimental diets in human subjects over a 2-week period (Garcia-Rojas, V., Maldonado- Hernandez, J., Mora-Escobedo, R., Izquierdo-Montalvo, G., Verma, M.P., Aragon-Aguilar, A., Angel-Serrato, D., Encarnacion-Fernandez, J.S. and Martin-Martinez, S., 2021. Use of13C-glucose breath test to assess treatment adherence in nutritional intervention trials. TIP. Revista Especializada en Ciencias Qulmico-Biologicas, 24). They concluded that 10 mg / day of13C-U6-glucose was the lower effective limit required to gradually increase the exhaled13CO2of a subject by a very modest level of +2%o. An important implication of this study is that it demonstrates that even small (e.g., 10 mg) daily doses of demonosaccharides will induce gradual enrichment of background13C in the body like that seen with the large doses of13C-lipid taggants discussed above. The cause for the accumulation of13C-derived from glucose taggants stems from the fact that the body readily converts the carbon atoms of exogenous glucose (and other carbohydrates) into other newly assembled molecules (e.g., fatty acids, and amino acids) thereby13C- enriching the lipid and protein pools in the body. This biochemical phenomenon is discussed in detail in scientific literature (McCue, M.D., Sivan, O., McWilliams, S.R. and Pinshow, B., 2010. Tracking the oxidative kinetics of carbohydrates, amino acids and fatty acids in the house sparrow using exhaled13CO2. Journal of Experimental Biology, 213(5), pp.782-789; McCue, M.D. and Pollock, E.D., 2013. Measurements of substrate oxidation using13CO2-breath testing reveals shifts in fuel mix during starvation. Journal of Comparative Physiology B, 183, pp.1039-1052). The present invention avoids the shortcoming of gradual13C-enrichment in the body by using13C-labeled taggants that are rapidly (e.g., within a few hours) cleared from the body.

[0016] An invention aimed at qualitatively characterizing (i.e. , low, medium, and high) adherence to a daily oral dosing regimen over a period of several weeks employs a stable isotopelabeled molecule (consisting of either a vitamin, protein, or enzyme) that can be periodically monitored from a blood sample of a subject (US20230233144A1). Unfortunately, this method is invasive (requiring a blood sample), and because the isotope-labeled taggant is not rapidly eliminated (like the13C-lipid and13C-carbohydrate taggants discussed above) it accumulates in the body and cannot be used to verify compliance on a daily basis. Moreover, because the body never reaches steady state equilibrium with the isotope of interest, it is not suitable for use over an extended period (e.g., months or years). The present invention differs from US20230233144A1 in that it does not involve any13C-labeled vitamins, proteins, or enzymes, and does not require blood sampling to track compliance.

[0017] Another group of solutions to directly monitor drug adherence involves combining therapeutic drugs with an additive that is enzymatically converted into either a volatile alcohol or other organic molecule that may be detected in a patient’s breath sample (US20070224128A1). The present invention differs from those methods in that CO2(which is not an organic molecule) is detected in the breath. A similar solution for compliance monitoring is a portable device that could either detect volatile organic compounds (mainly alcohols) or non-ordinary stable isotopes (preferably ‘Deuterium’ [2H] that is integrated into organic molecules) in the exhaled breath (WO2015134390A1). The present invention differs from these approaches in that it does not involve quantifying organic molecules or involve deuterium isotopes, the latter of which suffer from the potential of gradual bioaccumulation in the body as discussed above.

[0018] SUMMARY OF THE INVENTION

[0019] In one aspect, the present invention provides a pharmaceutical composition comprising a biologically active agent and one or more carbon 13 labelled taggant, wherein the taggant is a compound of formula (1): or a pharmaceutically acceptable salt thereof, wherein:

[0020] X is selected from: -OH, -O-(Ci to Ce alkyl), -O-(C2to Ce alkenyl), -O-(C2to Ce alkynyl), - O-(C3to Ce cycloalkyl), and -H; and

[0021] Y is selected from: -OH, -O-(Ci to Ce alkyl), -O-(C2to Ce alkenyl), -O-(C2to Ce alkynyl), - O-(C3to Ce cycloalkyl), and -Ci to C3alkyl wherein the Ci to C3alkyl group is optionally substituted with at least one hydroxy group, at least one primary amine group, and / or at least one oxo group; and wherein the compound of formula (1) contains at least one carbon 13 atom.

[0022] In a second aspect, the present invention provides a method of determining adherence to, or unauthorised use of, a pharmaceutical dosage regimen, the method comprising the steps of i) providing a pharmaceutical dosage form of one or more biologically active agent, wherein the pharmaceutical dosage form comprises one or more carbon 13 labelled taggant as defined herein; ii) measuring the level of carbon 13 containing CO2exhaled in one or more sample of the breath of a subject; iii) determining whether the subject has, or has not, consumed the pharmaceutical dosage form, within the window of activity of the taggant, by determining the level of carbon 13 containing CO2in the one or more sample as compared to a reference standard.

[0023] In a third aspect, the present invention provides a system for identifying use of a biologically active agent, the system comprising: i) a pharmaceutical dosage form of the biologically active agent, wherein the pharmaceutical dosage form comprises one or more carbon 13 labelled taggant as defined herein; ii) a means for collecting a sample of a subject’s breath; and iii) a means for measuring the amount of carbon 13 containing CO2in the sample.

[0024] In a fourth aspect, the present invention provides the use of a taggant as defined herein for preparing a composition as defined herein.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] Figure 1 is a graph depicting the delta over baseline (DOB) of13CO2in the breath of a subject vs time after ingestion of various13C-lipids and13C amino acids (e.g.,13C-glycine13C-leucine) at varying dosages, this figure has been bisected at 300 minutes in order to increase resolution;

[0027] Figure 2 is a graph depicting the DOB of13CO2in the breath of a subject vs time after ingestion of various13C-carbohydrates at varying dosages, this figure has been bisected at 300 minutes in order to increase resolution;

[0028] Figure 3 is a graph depicting the DOB of13CO2in the breath of a subject vs time after ingestion of13C-sodium bicarbonate at varying dosages;

[0029] Figure 4 is a graph depicting the per mil (%o) of13CO2in the breath of a subject vs time after ingestion of13C-sodium bicarbonate at varying dosages; Figure 5 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of13C-potassium carbonate at varying dosages;

[0030] Figure 6 is a graph depicting the %o of13CC>2 in the breath of a subject vs time after ingestion of13C-sodium carbonate at varying dosages;

[0031] Figure 7 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of13C-sodium formate at varying dosages;

[0032] Figure 8 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of sodium acetate-1-13C at varying dosages;

[0033] Figure 9 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of sodium acetate-2-13C at varying dosages;

[0034] Figure 10 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of acetic acid-1 -13C at varying dosages;

[0035] Figure 11 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of acetic acid-2-13C at varying dosages;

[0036] Figure 12 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of a combination of taggants of the present invention;

[0037] Figure 13 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of a combination of taggants of the present invention;

[0038] Figure 14 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of13C-potassium carbonate either formulated in a capsule or dissolved in aqueous solution;

[0039] Figure 15 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of sodium acetate-1-13C either formulated in a capsule or dissolved in aqueous solution; Figure 16 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of sodium bicarbonate-13C either formulated in a capsule or dissolved in aqueous solution; and

[0040] Figure 17 is a graph depicting the %o of13CO2in the breath of a subject vs time after ingestion of sodium bicarbonate-13C either formulated in a capsule or dissolved in aqueous solution.

[0041] DETAILED DESCRIPTION

[0042] Definition of Terms

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated by reference in their entirety.

[0044] Terms such as “13C”, “carbon 13”, “carbon 13 containing” and derivatives thereof are used interchangeably herein to refer to the carbon 13 isotope ora molecule comprising a carbon 13 isotope.

[0045] All numerical designations, e.g., pH, temperature, time, concentration, molecular weight, etc., including ranges, are approximations which are varied ( + ) or ( - ) by increments of, e.g., 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”, which is used to denote a conventional level of variability. For example, a numerical designation which is “about” a given value may vary by ± 10% of said value; alternatively, the variation may be ± 5%, ± 2%, or ± 1 % of the value. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. As used in the specification and claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

[0046] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, without excluding other elements. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any inessential significance for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this disclosure. Use of the term “comprising” herein is intended to encompass, and to disclose, the corresponding statements in which the term “comprising” is replaced by “consisting essentially of’ or “consisting of’.

[0047] A “subject,” refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, rodents, farm animals, sport animals, pets, and primates; for example murines, rats, rabbit, simians, bovines, ovines, porcines, canines, felines, equines, and humans. In a particular embodiment, the mammal is a human. In the context of the present invention, a “subject” may be the intended user of a biologically active agent, an intended non-user of a biologically active agent, a non-intended user of a biologically active agent, or a nonintended non-user of a biologically active agent.

[0048] “Administering” is defined herein as a means of providing an agent or a composition containing the agent to a subject in a manner that results in the agent being contacted with (e.g., being inside) the subject’s body. Such an administration can be by any route including, without limitation, oral, transdermal (e.g., by the vagina, rectum, or oral mucosa), by injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal, or into the central nervous system), or by inhalation (e.g., oral or nasal). Administration may also involve providing a substance or composition to a part of the surface of the subject’s body, for example by topical administration to the skin. Pharmaceutical preparations are, of course, given by forms suitable for each administration route.

[0049] “Treating” or “treatment” of a disease includes: (1) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a subject that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e. arresting or reducing the development of the disease or its clinical symptoms; and / or (3) relieving the disease, i.e. causing regression of the disease or its clinical symptoms.

[0050] An “effective amount” or “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depends upon a variety of factors including, for example, the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, the severity of the particular disorder being treated and the form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and / or in vivo tests initially can provide useful guidance on the proper doses for administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term “therapeutically effective amount” is an amount sufficient to treat (e.g., improve) one or more symptoms associated with the condition. The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein. In the context of the present invention, a “pharmaceutical composition” refers to a composition that is safe for human and / or animal consumption. In the context of the present invention, the “pharmaceutical composition” comprises a biologically active agent and one or more carbon 13 labelled taggant. Although the “pharmaceutical composition” may contain one or more pharmaceutically acceptable excipient, for example any one or more pharmaceutically acceptable excipient as described hereinbelow.

[0051] As used herein, the term “pharmaceutically acceptable excipient” encompasses any of the standard pharmaceutical excipients, for example as described in Remington’s Pharmaceutical Sciences (20th ed., Mack Publishing Co. 2000). Such excipients include binders, fillers, disintegrants, lubricants, glidants, solvents, diluents, casings, coating agents, acidity regulators, preservatives, stabilisers, thickening agents, flavourings, colourants, emulsifiers, solubilising agents, surfactants, buffers, or any combination thereof. For example, a binder may be hydroxypropyl methylcellulose (HPMC), polyvinylpyrrolidone (PVP), starch, or microcrystalline cellulose. For example, a diluent or filler may be lactose, dicalcium phosphate, mannitol, microcrystalline cellulose, or sucrose. For example, a disintegrant may be sodium starch glycolate, croscarmellose sodium, crospovidone, or pregelatinized starch. For example, a lubricant may be magnesium stearate, sodium stearyl fumarate, stearic acid, talc, or sodium lauryl sulphate. For example, a glidant may be silica gel, talc, or magnesium stearate. For example, a solvent or diluent may be water, ethanol, DMSO, or propylene glycol. For example, a casing or a coating agent may be gelatine, hydroxypropyl methylcellulose (HPMC), ethyl cellulose, polyvinyl alcohol, or shellac. For example, an acidity regulator may be citric acid, acetic acid, or phosphoric acid. For example, a preservative may be benzyl alcohol, parabens (e.g., methyl para ben, propylparaben), sodium benzoate. For example, a stabiliser may be ascorbic acid, sodium citrate, or citric acid. For example, a thickening agent may be xanthan gum, sorbitol, or carbomer. For example, a flavouring may be sucrose, sorbitol, artificial sweetener, or a flavouring oil. For example, a colourant may be iron oxide pigment, titanium dioxide pigment, or natural colourants (such as beet juice powder). For example, an emulsifier may be a polysorbate, sorbitan monostearate, or lecithin. For example, a solubilising agent may be propylene glycol, ethanol, or glycerol. For example, a surfactant may be sodium lauryl sulphate or a polysorbate. For example, a buffer may be a phosphate buffer (e.g., sodium phosphate), acetic acid / sodium acetate, citric acid / sodium citrate, tris (tris(hydroxymethyl)aminomethane), or boric acid / borate buffer. In the context of the present invention, a “biologically active agent” is any substance that exerts an effect on a subject. A “biologically active agent” may include any medicament, drug, contraceptive, hormone, enzyme, antibody, vitamin, peptide, vaccine, toxin, or antimicrobial.

[0052] In the context of the present invention, a “carbon 13 labelled taggant” is a compound or combination of compounds, each comprising at least one carbon 13 isotope (13C) within their molecular structure, which when consumed by a subject will be metabolised into carbon dioxide bearing a carbon 13 isotope (13CO2).

[0053] As used herein, the term “alkyl” means a saturated linear or branched free radical consisting essentially of carbon atoms and a corresponding number of hydrogen atoms. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, etc. Other alkyl groups will be readily apparent to those of skill in the art given the benefit of the present disclosure. The definition of “alkyl” also applies in the context of other groups which comprise alkyl groups, such as “-O(Ci-C3)alkyl”.

[0054] As used herein, the term “alkenyl” means a saturated linear or branched free radical consisting essentially of carbon atoms, a corresponding number of hydrogen atoms, and including one or more double carbon-carbon bond of either E or Z configuration unless specified. Exemplary alkenyl groups include ethenyl, n-propenyl, isopropenyl, etc. Other alkenyl groups will be readily apparent to those of skill in the art given the benefit of the present disclosure. The definition of “alkenyl” also applies in the context of other groups which comprise alkyl groups, such as “-O(Ci-C3)alkenyl”.

[0055] As used herein, the term “alkynyl” means a saturated linear or branched free radical consisting essentially of carbon atoms, a corresponding number of hydrogen atoms and one or more triple carbon-carbon bond. Exemplary alkynyl groups include ethynyl, n- propynyl, etc. Other alkynyl groups will be readily apparent to those of skill in the art given the benefit of the present disclosure. The definition of “alkynyl” also applies in the context of other groups which comprise alkyl groups, such as “-O(Ci-C3)alkynyl”.

[0056] As used herein, the term “cycloalkyl” means a saturated free radical having at least 3 to 6 carbon atoms (i.e., ring atoms) that form a ring. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The definition of “cycloalkyl” also applies in the context of other groups which comprise alkyl groups, such as “-O(Ci- C3)cycloalkyl”.

[0057] As used herein, the term “oxo” means a free radical wherein an oxygen atom is connected to the atom bearing this radical via a double bond. For example, where a carbon atom carries an oxo radical it forms a carbon-oxygen double bond. It will be appreciated that not all atoms within a given structure can be substituted by oxo, and that this will depend on the free valency of the atom to be substituted.

[0058] The compounds of the present disclosure are described, inter alia, by way of structural formulae. It will be appreciated that these formulae typically show only one form (e.g., resonance form, tautomeric form, etc.) of the compound, whereas certain compounds may exist in more than one such form. This will be readily apparent to the skilled reader. The present disclosure includes all possible tautomers of the compounds characterised by the structural formulae hereinbefore and below, including as single tautomers, or as any mixture of tautomers in any ratio.

[0059] The compounds of the disclosure may exist in the form of free acids or bases, or may exist as addition salts with suitable acids or bases. Methods for forming salts are described below and are also known in the art (see, e.g., Berge et al., J Pharm Sci. (1977) 66:1-19).

[0060] As used herein, the term “pharmaceutically acceptable” when used in connection with salts means a salt of a currently disclosed compound that may be administered without any resultant substantial undesirable biological effect(s) or any resultant deleterious interaction (s) with any other component of a pharmaceutical composition in which it may be contained.

[0061] Detailed Description of the Invention

[0062] In one aspect, the present invention provides a pharmaceutical composition comprising a biologically active agent and one or more carbon 13 labelled taggant, wherein the taggant is a compound of formula (1): or a pharmaceutically acceptable salt thereof, wherein:

[0063] X is selected from: -OH, -O-(Ci to Ce alkyl), -O-(C2to Ce alkenyl), -O-(C2to Ce alkynyl), - O-(C3to Ce cycloalkyl), and -H; and

[0064] Y is selected from: -OH, -O-(Ci to Ce alkyl), -O-(C2to Ce alkenyl), -O-(C2to Ce alkynyl), - O-(C3to Ce cycloalkyl), and -Ci to C3alkyl wherein the Ci to C3alkyl group is optionally substituted with at least one hydroxy group, at least one primary amine group, and / or at least one oxo group; and wherein the compound of formula (1) contains at least one carbon 13 atom.

[0065] When the pharmaceutical composition of the present invention is consumed by a subject, the carbon 13 labelled taggant will be metabolised into carbon 13 labelled carbon dioxide (13CO2). The resulting13CO2may then be detected in the breath of the subject. A level of13CO2which is significantly higher than a reference standard will confirm that the subject has consumed the pharmaceutical composition and thus the biologically active agent comprised therein. The reference standard may be, for example, a breath sample taken from the subject, or a different subject, taken in the absence of a carbon 13 labelled taggant, for example before beginning a dosing regimen. Alternatively, the reference standard may be a known reference standard for the background abundance of13CO2in biological systems, such as a reference standard for the background abundance of13CO2in a subject’s breath, for example a proposed global consensus value or a more targeted regional or population-specific value. Each taggant will have a different window of activity, which is defined as the period after consumption of the taggant in which a significantly elevated level of13CO2can be detected. The onset of the window of activity and the duration of the window of activity will depend on the pharmacokinetics of the taggant, i.e. the speed of its absorption and metabolism. The compounds of formula (1) are designed to have both a rapid onset of the window of activity, as well as a relatively short duration of window of activity. The advantage of this is that consumption or non-consumption of the taggant may be confirmed very rapidly after consumption or non-consumption. Additionally, a relatively short duration of window of activity allows for greater compatibility with a regular dosage regimen, such as once daily dosing, or more multiple daily dosing, such as twice daily, thrice daily, four times daily, five times daily, or six times daily. This is because a short window of activity allows for a return of the subject’s breath13CO2level to baseline before the next dosage. This prevents any build up or accumulation of taggant in the subject which may interfere with subsequent breath samples. The pharmaceutical composition comprises one or more taggant and a biologically active agent. Consumption of the pharmaceutical composition will therefore entail consumption of both the taggant and the biologically active agent. Therefore, the pharmaceutical composition is useful for determining whether a subject has or has not consumed biologically active agent. The pharmaceutical composition is therefore useful for determining adherence to, or non-adherence to, a dosage regimen of the biologically active agent. As discussed above, patient compliance may be a problem for many drug dosage regimens, so it is useful to be able to confirm whether a subject has indeed complied with a dosage regimen. Similarly, consumption of the biologically active agent by an unintended user can also be confirmed. Many biologically active agents may be desirable to consume by unauthorised persons, e.g. a subject who is not the intended recipient, which may be the case for drugs of abuse or performance enhancing drugs. According to the present invention, a biologically active agent having abuse potential may be formulated into a pharmaceutical composition comprising one or more taggant of the present invention so that unauthorised use may be detected.

[0066] The taggant is a compound of formula (1) as defined herein. Formula (1) comprises a carbonyl group with two substituents which may form, various carbonyl containing functional groups, such as an aldehyde, a carboxylic acid or carboxylate salt, a carbonic acid or carbonate salt, or alkyl, alkenyl, alkynyl, and cycloalkyl carbonates, although carboxylic acids or carboxylate salts, carbonic acids or carbonate salts are preferred. These structures are rapidly metabolised by the body into CO2. In a preferred embodiment, the taggant is a compound of formula (1): or a pharmaceutically acceptable salt thereof, wherein:

[0067] X is selected from: -OH, and -H; and

[0068] Y is selected from: -OH, and -Ci to C3 alkyl wherein the Ci to C3 alkyl group is optionally substituted with at least one hydroxy group, at least one primary amine group, and / or at least one oxo group; and wherein the compound of formula (1) contains at least one carbon 13 atom. More preferably, X is: -OH. More preferably Y is selected from: -OH, and -Ci to C2alkyl wherein the Ci to C2alkyl group is optionally substituted with at least one hydroxy group, at least one primary amine group, and / or at least one oxo group; or Y is selected from: -OH, and -Ci alkyl wherein the Ci alkyl group is optionally substituted with at least one hydroxy group, at least one primary amine group, and / or at least one oxo group.

[0069] Preferably the Ci to C3 alkyl group, Ci to C2alkyl group or -Ci alkyl group is optionally substituted with at least one hydroxy group, and / or at least one oxo group, or more preferably is not substituted.

[0070] For example, wherein X and Y are both -OH groups, then the taggant is carbonic acid. Wherein X is -H and Y is an -OH group, then the taggant is formic acid. Wherein X is an - OH group and Y is a -Ci to C3 alkyl group, then the taggant is a carboxylic acid, such as acetic acid or propionic acid. Wherein X is -H and Y is a -Ci to C3 alkyl group, then the taggant is an aldehyde, such as acetaldehyde or propionaldehyde. Certain carboxylic acids or aldehydes may be substituted with at least one hydroxy group, at least one primary amine group, and / or at least one oxo group. For example, substitution with a hydroxy group may provide lactic acid or glycolaldehyde, substitution with an amine group may provide glycine, substitution with an oxo group may provide pyruvic acid or acetoacetic acid. Substitution with a hydroxy group and an oxo group may provide oxalic acid. Substitution with two hydroxy groups may provide glyceraldehyde. Substitution with three hydroxy groups and two oxo groups may provide tartaric acid.

[0071] Some particularly preferred taggants include carbon 13 labelled lactic acid, pyruvic acid, carbonic acid, formic acid, acetic acid, or a pharmaceutically acceptable salt thereof. For example, a carboxylate salt, a carbonate salt, or a bicarbonate salt. Whilst any pharmaceutically acceptable salt form of a compound of formula (1) may be used, sodium and potassium salts are preferred, such as sodium carboxylates, potassium carboxylates, potassium carbonate, sodium carbonate, sodium bicarbonate, or potassium bicarbonate. A carbonic acid salt may be a mono-base addition salt, such as sodium bicarbonate or potassium bicarbonate, or a di-base addition salt, such as sodium carbonate or potassium carbonate.

[0072] The compound of formula (1) must contain at least one carbon 13 atom. For avoidance of doubt, at least one carbon 13 atom must be contained within formula (1 ) itself as depicted above and not just within the counter-ion of a salt form of a compound of formula (1). Nevertheless, a salt form may comprise one or more carbon 13 atoms, so long as at least one carbon 13 atoms is comprised within the compound of formula (1). The compound of formula (1) may contain multiple carbon 13 atoms, such as 2, 3, 4, or more carbon 13 atoms. Optionally, all of the carbon atoms in the compound of formula (1) are carbon 13 atoms. It is preferred that the carbonyl carbon depicted in formula (1) is a carbon 13 atom, as this carbon is particularly labile to metabolism to CO2. In this case, the compound of formula (1) has the following structure: wherein X and Y are as defined in the first aspect defined hereinabove or in any embodiment disclosed in connection therewith.

[0073] The pharmaceutical composition may comprise multiple taggants of formula (1), for example two taggants of formula (1), or three taggants of formula (1). This allows for combination of the beneficial features of multiple taggants, for example by fine tuning the window of activity. For example, a taggant with a more rapid onset of window of activity may be combined with a taggant having a longer duration of window of activity in order to provide a combination of taggants which provides both a rapid onset and the desired duration of window of activity. Carbonic acid and carbonate salt taggants tend to have relatively more rapid onsets and shorter durations of window of activity, whereas carboxylic acid and carboxylate salt taggants tend to have relatively less rapid onsets and longer durations of window of activity. The pharmaceutical composition may comprise a carboxylic acid containing compound of formula (1) or a pharmaceutically acceptable salt thereof, and a carbonic acid of formula (1) or a pharmaceutically acceptable salt thereof.

[0074] The pharmaceutical composition may be formulated for administration to a subject via any suitable route of administration, for example, the pharmaceutical composition is formulated for oral administration, transdermal administration, vaginal administration, rectal administration, transmucosal administration, injection (e.g. subcutaneous injection, intravenous injection, or intramuscular injection), or for inhalation (e.g. oral inhalation or nasal inhalation); preferably wherein the pharmaceutical composition is formulated for oral administration or intravenous injection. Typically, the pharmaceutical composition is formulated forthe same route of administration as intended forthe biologically active agent contained therein.

[0075] Typically, the pharmaceutical composition will be formulated in a dosage form comprising a single dosage of the taggant and a single dosage of the biologically active agent, such that a user may ingest a dosage of taggant alongside each dosage of biologically active agent. The dosage of the biologically active agent may be any suitable dosage as is required by a desired dosage regimen. The dosage of the taggant will typically be a dosage suitable to provide a window of activity of the taggant which will conclude before the subsequent scheduled dosage takes place. In other words, the dosage of the taggant is such that the resulting13CO2level in the subject’s breath is significantly elevated above the baseline as defined by a reference standard after consumption of the pharmaceutical composition, and such that the resulting13CO2level in the subject’s breath is no longer significantly elevated above the baseline as defined by a reference standard before the subsequent scheduled dosage. In this way, the consumption of each scheduled dosage by the subject can be confirmed without any previously consumed taggant impacting the13CO2level in the breath sample.

[0076] Typically, the window of activity may continue for a period of 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240 minutes, 270 minutes, 300 minutes, 330 minutes, 360 minutes, 390 minutes, 420 minutes, 450 minutes, 480 minutes, 510 minutes, 540 minutes, 570 minutes, or 600 minutes after consumption of the taggant. Typically, the window of activity may begin a period of 30 seconds, 60 seconds, 90 seconds, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, or 15 minutes after consumption of the taggant.

[0077] More rapidly metabolised taggants provide the advantage of a quicker onset of the window of activity. More rapidly metabolised taggants provide a shorter duration of the window of activity which may be more suited to dosage regimens with more frequent dosing. Less rapidly metabolised taggants provide a longer duration of the window of activity which may be more suited to dosage regimens with less frequent dosing. A rapid onset and longer duration of the window of activity can be achieved using a pharmaceutical composition comprising both a rapidly metabolised taggant and a less rapidly metabolised taggant. Carbonate and bicarbonate salts are typically more rapidly metabolised, whilst carboxylic acids and carboxylates are relatively less rapidly metabolised. Taggants wherein the carbonyl carbon depicted in formula (1) is a13C atom will typically be more rapidly metabolised than taggants where the13C atom is comprised with the X or Y group depicted in formula (1). A larger dosage of taggant will typically provide a quicker onset of the window of activity and a longer duration of the window of activity. The formulation of the pharmaceutical composition may also impact the window of activity, for example, a faster acting route of administration such as an intravenous formulation will quicken the onset of the window of activity and decrease the duration of the window of activity. A slower acting route of administration such as an oral formulation will slow the onset of the window of activity and increase the duration of the window of activity. As would be appreciated, there are numerous ways to fine tune the window of activity as desired to correspond to the desired dosage regimen of the biologically active agent.

[0078] For example, the pharmaceutical composition may comprise from 1 mg to 200 mg, 5 mg to 100 mg, 10 mg to 75 mg, 10 mg to 50 mg of the one or more carbon 13 labelled taggant. The pharmaceutical composition may comprise from 1 mg to 200 mg, 5 mg to 200 mg, 10 mg to 200 mg, 20 mg to 200 mg, 30 mg to 200 mg, 40 mg, to 200 mg, or 50 mg to 200 mg of the one or more carbon 13 labelled taggant. The pharmaceutical composition may comprise from 1 mg to 200 mg, 1 mg to 150 mg, 1 mg to 100 mg, 1 mg to 80 mg, 1 mg to 70 mg, 1 mg to 60 mg, 1 mg to 50 mg, 1 mg to 40 mg, or 1 mg to 30 mg of the one or more carbon 13 labelled taggant. For example, the pharmaceutical composition may comprise from 0.01 mg / kg to 2 mg / kg, 0.05 mg / kg to 1.5 mg / kg, 0.1 mg / kg to 1.0 mg / kg, or 0.15 to 0.75 mg / kg.

[0079] The biologically active agent may be a drug, such as an antibiotic, an antihypertensive, a statin, insulin, metformin, an antidepressant, an antiretroviral, an antipsychotic, an immunosuppressant, a drug of abuse, an anti-epileptic, a proton pump inhibitor, a contraceptive, an anticoagulant, an opioid, a psychomotor stimulant, a chemotherapy drug, a hypertension management drug, a psychotropic drug, a sedative hypnotic, and any combination thereof. The active agent may be a drug which has negative side effects and thus risks poor patient compliance. The breath of a subject who was intended to use the drug may be tested to confirm that the drug has or has not been used.

[0080] The biologically active agent may be a drug of abuse, for example, a controlled or prescription drug which has abuse potential, or an illegal drug. The drug of abuse may be intended for consumption by a subject, such as a patient or clinical trial subject, but risk unauthorised consumption by an unintended subject. Alternatively, the drug of abuse may not be intended for consumption, and may be for example, intended for use in research, as a reference standard, or in sniffer dog training. The breath of a subject who was not intended to use the drug may be tested to confirm that the drug has or has not been used.

[0081] In a second aspect, the present invention provides a method of determining adherence to, or unauthorised use of, a pharmaceutical dosage regimen, the method comprising the steps of i) providing a pharmaceutical dosage form of one or more biologically active agent, wherein the pharmaceutical dosage form comprises one or more carbon 13 labelled taggant as defined herein; ii) measuring the level of carbon 13 containing CO2exhaled in one or more sample of the breath of a subject; iii) determining whether the subject has, or has not, consumed the pharmaceutical dosage form, within the window of activity of the taggant, by determining the level of carbon 13 containing CO2in the one or more sample as compared to a reference standard.

[0082] Sampling the breath of a subject may either confirm consumption of the pharmaceutical dosage form in the case that the breath sample contains a significantly higher level of carbon 13 containing CO2than the reference standard or confirm non-consumption of the pharmaceutical dosage form within the window of activity in the case that the breath sample does not contain a significantly higher level of carbon 13 containing CO2than the reference standard. The subject whose breath is tested may or may not be the intended user of the pharmaceutical dosage form. Where the breath of the intended user is tested, a positive result may determine use as intended, i.e. adherence to the pharmaceutical dosage regimen, or a negative result may determine failure to use the pharmaceutical dosage regimen as intended, i.e. non-adherence. Where the breath of a subject who is not the intended user of the pharmaceutical dosage regimen is tested, a positive result may determine unauthorised use of the pharmaceutical dosage form, or a negative result may determine non-use use of the pharmaceutical dosage form during the window of activity. Furthermore, the method may be used to detect ‘excessive’ use of a biologically active agent in a subject who is the intended user of the biologically active agent. For example, an intended dosage of a biologically active agent, such as a drug, may be combined with an amount of taggant that is known to produce an expected response, for example a known dose of taggant can be expected to produce a relatively consistent DOB or %o of13CO2in the breath of the subject. Additionally, an initial calibration reading can also be taken from the subject after consumption of the known dose of taggant. A larger than expected response, for example a larger than expected DOB or %o of13CO2in the breath of the subject, can indicate that a larger than intended dosage of the biologically active agent has been consumed. This method may be particularly useful with prescription drugs that have a high abuse potential.

[0083] In the context of the present invention, the reference standard refers to the level of carbon 13 containing CO2that would normally be expected in a subject’s breath in the absence of any carbon 13 labelled taggant, or metabolites thereof. Typically, about 1.1% of the carbon in carbon dioxide in human breath is carbon 13, which is similar to the natural abundance of carbon 13 in the atmosphere. However, the exact percentage can vary slightly based on individual metabolic processes and dietary factors. As the taggants of the present invention give rise to very large delta over baseline (DOB) readings of carbon 13 containing CO2any variations due to idiosyncrasy or diet will not significantly impact the results. Therefore, it is not necessary that a reference standard be measured from the same subject. The reference standard may simply be the natural abundance of carbon 13 in the atmosphere. The skilled person would also have access to expected reference standards from standard medical textbooks.

[0084] Nevertheless, a reference sample may optionally be taken from a subject, preferably the subject to be tested in the absence of the taggant, and this reference sample used as the reference standard. The method may further comprise the step of collecting a sample of the breath of the subject for use as the reference standard, preferably the method may further comprise the step of collecting multiple samples of the breath of the subject at differing time points, for example 2, 3, 4, 5, or more samples, for use as the reference standard. Graphical representation may be used to compare one or more samples to multiple reference standards.

[0085] Determining the level of carbon 13 containing CO2exhaled in the breath of the subject may be performed using any suitable method of detecting carbon 13 containing CO2, such as, use ofan isotope analyser, FTIR-Fourier transform infrared spectroscopy, conventional isotope ratio mass spectrometry, non-dispersive infrared spectrometry, or laser-based spectrometry, such as cavity ring down spectroscopy, and off-axis integrated cavity output spectroscopy. For example, non-dispersive infrared spectroscopy may be used with a Helifan (RTM) Plus analyser paired with a Fan-AS (RTM) Autosampler, conventional isotope ratio mass spectrometry may be used with a Sercon (RTM) ABCA2 breath testing device, and laser-based spectroscopy may be used with an ABB (RTM) GLA132-CCIA2 device.

[0086] A breath sample may be analysed for carbon 13 containing CO2immediately, or the method may comprise temporarily storing one or more sample of the breath of the subject in a sealed container for future determination of the level of carbon 13 containing CO2in the one or more sample. As would be appreciated, carbon 13 is a stable isotope, and as such a sealed container will allow for storage of the breath sample indefinitely. Detection of the level of carbon 13 containing CO2in the stored sample will therefore give a measure of the level of carbon 13 containing CO2in the breath of the subject at the time that the sample was taken. Alternatively, a subject may exhale directly into the intake port of a means for measuring the amount of carbon 13 containing CO2in the sample, such as those discussed above, so that a near-real-time measurement can be made. This is well suited to an on premises / at point of care approach where there is no requirement for sample storage.

[0087] The pharmaceutical dosage form which is useful in the method of the present invention may be prepared by combining the one or more biologically active agent with the one or more carbon 13 labelled taggant. The method of the present invention may further comprise the preceding step of preparing the pharmaceutical dosage form by combining the one or more biologically active agent with the one or more carbon 13 labelled taggant, optionally with one or more pharmaceutically acceptable excipient as described herein.

[0088] The pharmaceutical dosage form may be formulated for administration via any suitable route of administration, for example the pharmaceutical dosage form may be formulated may be formulated for administration as described in relation to the first aspect, or any embodiment described in relation thereto. The biologically active agent may be as described in relation to the first aspect, or any embodiment described in relation thereto.

[0089] In a third aspect, the present invention provides a system for identifying use of a biologically active agent, the system comprising: i) a pharmaceutical dosage form of the biologically active agent, wherein the pharmaceutical dosage form comprises one or more carbon 13 labelled taggant as defined herein; ii) a means for collecting, and optionally storing, a sample of a subject’s breath; and iii) a means for measuring the amount of carbon 13 containing CO2in the sample.

[0090] The pharmaceutical dosage form, biologically active agent, and / or carbon 13 labelled taggant may be as described in relation to the first aspect, or any embodiment described in relation thereto.

[0091] The means for collecting a sample of a subject’s breath may be any standard breath collection apparatus known in the art. The step of breath collection for analytic purposes is within the capabilities of the skilled person. The means for collecting a sample of a subject’s breath may comprise a means for storing a sample of a subject’s breath. The means for storing a sample of a subject’s breath may be any suitable container that is capable of being hermetically sealed, such as a glass breath collection vial. The means for measuring the amount of carbon 13 containing CO2in the sample may be any device capable of measuring the carbon 13 containing CO2, for example, any device capable of any of the carbon 13 containing CO2detection methods described in relation to the second aspect. For example, the means for measuring the amount of carbon 13 containing CO2in the sample may be a Helifan (RTM) Plus analyzer paired with a Fan-AS (RTM) Autosampler.

[0092] In a fourth aspect, the present invention provides the use of a taggant as defined herein for preparing a composition as defined herein.

[0093] The invention will now be described by reference to the following non-limiting Examples and the Figures.

[0094] EXAMPLES

[0095] Ingestion of dissolved13C-taggants

[0096] 13C-labled chemicals (98-99% pure) were obtained from commercial sources. Tracer doses ranging in mass from 5 mg to 100 mg were measured to ±0.1 mg and dissolved in 10ml of water. In some cases, two different13C-labeled taggants with different kinetic properties were ingested simultaneously to document the additive effects.

[0097] The solutions were ingested by a 72kg male (age 50 years) subject between 8:00am and 10:00am. Each ingested 10ml solution was followed by ingestion of 30ml of water to facilitate delivery to the stomach. Baseline breath samples were collected at three timepoints (e.g., typically -10, -5, and 0 minutes) prior to ingestion by exhaling through a plastic straw into a 12ml glass breath collection vial that was immediately capped thereafter. Following dosing breath samples were periodically collected (using the same vials method as the baseline samples) over the subsequent 5 to 10 hours depending on the13C-tracer used.

[0098] All breath samples were analyzed for13CO2levels (i.e., the change in13CO2) within 48 hours of collection using non-dispersive infrared (NDIR) spectroscopy by a Helifan (RTM) Plus analyzer paired with a Fan-AS (RTM) Autosampler according to standard published protocols (Balakrishnan, B., Yan, X., McCue, M.D., Bellagamba, O., Guo, A., Winkler, F., Thall, J., Crawford, L., Dimen, R., Chen, S. and McEnaney, S., 2024. Whole-body galactose oxidation as a robust functional assay to assess the efficacy of gene-based therapies in a mouse model of Galactosemia. Molecular Therapy Methods & Clinical Development, 32(1)., 2024.; Twining, C.W., Shipley, J.R., McCue, M.D., Pokrovsky, I., Gregoire, A., Faivre, B., Wikelski, M. and Partecke, J., 2023. Energetics and fuel use vary with migration strategy across populations of Common Blackbirds. Functional Ecology, 37(7), pp.1910-1921.). It should be noted that13CO2breath samples collected in this manner are known to remain stable and accurate for several weeks (McCue, M.D., Sivan, O., McWilliams, S.R. and Pinshow, B., 2010. Tracking the oxidative kinetics of carbohydrates, amino acids and fatty acids in the house sparrow using exhaled13CO2. Journal of Experimental Biology, 213(5), pp.782-789.).

[0099] Delta over baseline (DOB) values, a standard measure of13C-tracer-induced enrichment of13CO2levels in the breath, were calculated by comparing post-dose 513CO2at each time point to the average 513CO2obtained from the respective baseline measurements. Clinically ‘meaningful’ elevations in DOB are interpreted as values greater than +5%o like the critical values used in standard13C-Urea breath testing for Helicobacter pylori infections (Gisbert, J.P. and Pajares, J.M., 2004.13C-urea breath test in the diagnosis of Helicobacter pylori infection-a critical review. Alimentary Pharmacology & Therapeutics, 20(10), pp.1001-1017.). While it is a standard practice to compare a patient’s baseline breath sample with a postdosing breath sample (e.g., EP1018938A2; US665612B1 , this approach is based on the fact that the maximal b13CO2-enrichment caused by many13C-tracers tends to be modest (e.g., +10%o). As described elsewhere in this invention, more conservative ‘critical’ DOB values (e.g., +10%o) based on population norms (Tanis, A.A., Rietveld, T., Van Den Berg, J.W.O., Wattimena, J.D. and Swart, G.R., 2000. Influence of the13C-enrichment of the habitual diet on a13CO2breath test used as an index of liver glycogen oxidation: a validation study in western Europe and Africa. Nutrition, 16(1), pp.6-10.) could be used if baseline b13CO2of a patient were not collected or are otherwise unknown. Figures 3 to 6 show that as little as 10mg of some13C-taggants (e.g.,13C-Na-Bicarbonate) can cause the b13CO2in the breath to become more than +20%o enriched, thereby precluding the need for an actual baseline measurement for accurate determinations.

[0100] 13C-Taggant dose responses were also examined to characterize the effects of dose and magnitude and duration of detectable responses. For the small molecule taggants preferred in this invention doses ranged from 5mg (69 ug / kg) to 100mg (1390 ug / kg). Larger doses were needed to elicit meaningful13CO2enrichments for13C-lipid,13C-amino acid, and13C-carbohydrate taggants (Figures 1 and 2) because of their lower potency.

[0101] Figures 3 to 8 and 10 show the window of activity of preferred taggants of the present invention at various dosages. As can be seen, the window of activity for these taggants has a rapid onset and a relatively short duration. A significant elevation in13CO2was achieved even with relatively small taggant dosages, such as 10 mg. Figures 9 and 11 show the window of activity of activity of taggants of formula (1) wherein the13C atom is not the carbonyl carbon, Figures 8 and 10 show the comparative taggants (i.e. the same molecule) where the13C atom is the carbonyl carbon. Taggants of formula (1) wherein the13C atom is not the carbonyl carbon tend to provide a smaller elevation in13CO2but a longer duration of window of activity.

[0102] Depending on the desired13CO2outcome it is possible to use multiple13C-taggants to optimize initial response times and duration. For example, Figure 12 illustrates that a mixture of 10mg K-13C-Bicarbonate and 20 mg Na-13C-Acetate-2 generate an additive response that may be more desirable than either taggant when used individually yielding a fast initial response that occurs within two minutes and a duration where DOB remains +10%o for 30 minutes). Similarly, Figure 13 illustrates that a mixture of 10 mg Na-13C- Bicarbonate and 10 mg Na-13C-Acetate-1 generate an additive response that may be more desirable than either taggant when used individually yielding a fast initial response that occurs within two minutes and a duration where DOB remains +10%o for 30 minutes).

[0103] Those with access to13CO2monitoring devices that are capable of real-time measurements (especially CRDS, ICOS (RTM), and other laser-based technologies) will be able to use this approach to continuously characterize13CO2production in subjects over several minutes to hours. These data would allow determinations of time-dependent13CO2profiles and permit users to further examine area under the curve (AUC) responses and detect even smaller13CO2-enrichments than a single post-dose measurement might allow.

[0104] Tests were conducted using13C-taggents ingested in capsule form to compare differences in time course (e.g., onset and duration) and magnitude (e.g., peak DOB values) with the dissolved chemical taggants. In this case the same protocol (as described above for dissolved chemicals) was used except that the13C-tracerwas placed into a size 1 gelatin capsule and swallowed with 30 ml of water. For example, Figures 14 to 17 depict the difference in the window of activity which can be seen from the %o of13CO2in the breath of a subject vs time after ingestion of taggants which are either formulated in a capsule or dissolved in aqueous solution. As can be seen, in all cases, formulation of the taggant in a capsule slightly delayed the onset of the window of activity as compared to formulation of the taggant in aqueous solution.

Claims

CLAIMS:

1. A pharmaceutical composition comprising a biologically active agent and one or more carbon 13 labelled taggant, wherein the taggant is a compound of formula (1):or a pharmaceutically acceptable salt thereof, wherein:X is selected from: -OH, -O-(Ci to Ce alkyl), -O-(C2to Ce alkenyl), -O-(C2to Ce alkynyl), -O-(C3 to Ce cycloalkyl), and -H; andY is selected from: -OH, -O-(Ci to Ce alkyl), -O-(C2to Ce alkenyl), -O-(C2to Ce alkynyl), -O-(C3 to Ce cycloalkyl), and -Ci to C3 alkyl wherein the Ci to C3 alkyl group is optionally substituted with at least one hydroxy group, at least one primary amine group, and / or at least one oxo group; and wherein the compound of formula (1) contains at least one carbon 13 atom.

2. The pharmaceutical composition of claim 1 , wherein:X is selected from: -OH, and -H; andY is selected from: OH, and -Ci to C3 alkyl wherein the Ci to C3 alkyl group is optionally substituted with at least one hydroxy group, at least one primary amine group, and / or at least one oxo group.

3. The pharmaceutical composition of claim 1 or claim 2, wherein the one or more taggant comprises or consists of at least two different compounds of formula (1), preferably wherein the one or more taggant comprises or consists of a carboxylic acid containing compound of formula (1) or a pharmaceutically acceptable salt thereof, and a carbonic acid of formula (1) or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition of any one of the preceding claims, wherein the one or more taggant comprises or consists of a carbon 13 labelled lactic acid, pyruvic acid, carbonic acid, formic acid, acetic acid, or a pharmaceutically acceptable salt thereof; preferably a carbonate salt or a bicarbonate salt, such as potassium carbonate, sodium carbonate, sodium bicarbonate, or potassium bicarbonate.

5. The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition comprises from 5 mg to 100 mg of the one or more carbon 13 labelled taggant, preferably from 10 mg to 75 mg.

6. The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition is formulated for oral administration, transdermal administration, vaginal administration, rectal administration, transmucosal administration, injection (e.g. subcutaneous injection, intravenous injection, or intramuscular injection), or for inhalation (e.g. oral inhalation or nasal inhalation); preferably wherein the pharmaceutical composition is formulated for oral administration or intravenous injection.

7. The pharmaceutical composition of any one of the preceding claims, wherein the biologically active agent is selected from: an antibiotic, an antihypertensive, a statin, insulin, metformin, an antidepressant, an antiretroviral, an antipsychotic, an immunosuppressant, a drug of abuse, an anti-epileptic, a proton pump inhibitor, a contraceptive, an anticoagulant, an opioid, a psychomotor stimulant, a chemotherapy drug, a hypertension management drug, a psychotropic drug, a sedative hypnotic, and any combination thereof.

8. A method of determining adherence to, or unauthorised use of, a pharmaceutical dosage regimen, the method comprising the steps of i) providing a pharmaceutical dosage form of one or more biologically active agent, wherein the pharmaceutical dosage form comprises one or more carbon 13 labelled taggant as defined in any one of claims 1 to 5; ii) measuring the level of carbon 13 containing CO2exhaled in one or more sample of the breath of a subject; iii) determining whether the subject has, or has not, consumed the pharmaceutical dosage form, within the window of activity of the taggant, by determining the level of carbon 13 containing CO2in the one or more sample as compared to a reference standard.

9. The method of claim 8, wherein determining the level of carbon 13 containing CO2exhaled in the breath of the subject is performed using FTIR-Fourier transform infrared spectroscopy, isotope ratio mass spectrometry, nondispersive infrared spectrometry, or laser-based spectrometry, such as cavity ring down spectroscopy.

10. The method of claim 8 or claim 9, wherein the method comprises temporarily storing one or more sample of the breath of the subject in a sealed container for future determination of the level of carbon 13 containing CO2in the one or more sample.11 . The method of any one of claims 8 to 10, wherein the method comprises the preceding step of preparing the pharmaceutical dosage form by combining the one or more biologically active agent with the one or more carbon 13 labelled taggant, optionally with one or more pharmaceutically acceptable excipient.

12. The method of any one of claims 8 to 11 , wherein the method further comprises the step of collecting a sample of the breath of the subject for use as the reference standard.

13. The method of any one of claims 8 to 12, wherein the pharmaceutical dosage form is as defined in claim 6.

14. The method of any one of claims 8 to 13, wherein the biologically active agent is as defined in claim 7.

15. A system for identifying use of a biologically active agent, the system comprising: i) a pharmaceutical dosage form of the biologically active agent, wherein the pharmaceutical dosage form comprises one or more carbon 13 labelled taggant as defined in any one of claims 1 to 5; ii) a means for collecting, and optionally storing, a sample of a subject’s breath; and iii) a means for measuring the amount of carbon 13 containing CO2in the sample.

16. The system of claim 15, wherein the pharmaceutical dosage form is as defined in claim 6.

17. The system of claim 15 or claim 16, wherein the biologically active agent is as defined in claim 7.

18. Use of a taggant as defined in any one of claims 1 to 5 for preparing a composition as defined in any one of claims 1 to 7.

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