Apparatus and methods for assessing drug tissue distribution

Abstract

A method for determining a tissue distribution phenotype of an animal or a human subject for an exogenous agent, such as a drug. The method includes administering the exogenous agent to the subject, and at one or more time points after administration, determining an amount of the exogenous agent in the interstitial fluid of a tissue of the subject. The amount of exogenous agent may be determined by an electrochemical aptamer-based sensor. The determined amount is used on its own or in conjunction with another value to determine a tissue distribution phenotype of the subject for the exogenous agent.

Description

[0001] APPARATUS AND METHODS FOR ASSESSING DRUG TISSUE DISTRIBUTION

[0002] FIELD

[0003]

[0001] , The present disclosure relates generally to the field of prophylaxis and therapy of animal subjects by way of drug administration. More particularly, the disclosure is directed to the determination of a drug tissue distribution phenotype for a subject. Also provided are methods for assessing the tissue availability of a drug, and also the extent to which a tissue is perfused.

[0004] BACKGROUND

[0005]

[0002] , Modern medicine provides a myriad of drugs having efficacies in the treatment or prevention of a broad range of conditions.

[0006]

[0003] , Typically, a drug is administered to a subject according to a dosage regime with the aim of achieving at least a minimum efficacious amount in the subject. A drug may be administered over a period of time by way of continuous infusion or repeat discrete administrations over a period of time for which exposure to the drug is required.

[0007]

[0004] , Some drugs have a low therapeutic index, having the propensity to cause toxic side effects. Accordingly, dosing is set such that excursion over a maximum amount is prevented. Some drugs have a high therapeutic index, presenting little danger of toxicity even where dosed at levels significantly higher than that required for efficacy.

[0008]

[0005] , Drugs are typically monitored for minimum and / or maximum by reference to concentration in the blood. Where drug monitoring is indicated, blood may be drawn from a vein of the subject at intervals, with each sample being dispatched to an analytical laboratory

[0009]

[0006] , Even with careful monitoring, some drugs are dosed at a level that is not efficacious, not maximally efficacious, toxic, or with wastage. Suboptimal dosing can occur even when blood samples shows that the drug is at a level that is appropriate.

[0010]

[0007] , Even where a drug is not considered overly toxic, the dosage required to exert a desired therapeutic or prophylactic effect can be difficult to achieve. For example, some subjects extensively metabolise a drug in the liver via the cytochrome P450 enzyme system thereby rapidly diminishing the amount of active drug in circulation. In such cases, higher dosages or more frequent dosing may be required to achieve the required efficacy. Other subjects may metabolise drug to only to a limited extent, thereby requiring lower dosages or less frequent dosing.

[0011]

[0008] , Drug screening and rational drug design aim to identify or generate agents that are resistant to metabolism by the liver, or in some other way are able to achieve useful concentrations for extended periods in a target tissue. There is little point in designing a drug having a very high level of cell receptor binding, for example, if the drug cannot accumulate to any level useful concentration about the receptor. Those concerned with the design and screening of new drugs cannot easily assess candidate drugs for an ability to reach target tissues.

[0012]

[0009] , Separate to the problems stated above, various sensors have been devised to detect the amount of drug in the interstitial fluid of a subject. It is presently unknown as to what type of calibration is required (if any) such that sensor output is clinically meaningful.

[0013]

[0010] , It is an aspect of the present disclosure to provide an improvement or an alternative to the prior art.

[0014] [Oi l], The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present disclosure. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0015] SUMMARY

[0016]

[0012] , In a first aspect, but not necessarily the broadest aspect, there is provided a method for determining a tissue distribution phenotype of an animal or a human subject for an exogenous agent, the method comprising the steps of: administering the exogenous agent to the subject, at one or more time points after administration, determining an amount of the exogenous agent in the interstitial fluid of a tissue of the subject, wherein the determined amount is used on its own or in conjunction with another value to determine a tissue distribution phenotype of the subject for the exogenous agent.

[0013] , In one embodiment of the first aspect, the method comprises the steps of: applying to a tissue of the subject an electrochemical sensor based on a recognition element configured to specifically detect the exogenous agent such that a working electrode of the sensor contacts an interstitial fluid of the tissue, wherein the determination of an amount of the exogenous agent in the interstitial fluid is by made by reference to an output of the sensor.

[0017]

[0014] , In one embodiment of the first aspect, the determined amount is compared with an amount of the exogenous agent in the systemic circulation of the subject (i) determined contemporaneously or otherwise by assay, or (ii) predicted, inferred, or estimated.

[0018]

[0015] , In one embodiment of the first aspect, the tissue distribution phenotype relates to the extent to which, or the rate at which, the exogenous agent distributes from the systemic circulation to the tissue.

[0019]

[0016] , In one embodiment of the first aspect, the tissue distribution phenotype is a qualitative or a quantitative value.

[0020]

[0017] , In one embodiment of the first aspect, the sensor comprises a working electrode that penetrates the skin so as to contact the interstitial fluid of the tissue.

[0021]

[0018] , In one embodiment of the first aspect, the working electrode is a needle, a microneedle, or a wire that extends into the subcutaneous tissue of the subject.

[0022]

[0019] , In one embodiment of the first aspect, the exogenous agent is a therapeutic agent or a prophylactic agent.

[0023]

[0020] , In one embodiment of the first aspect, the exogenous agent is a molecule that is potentially capable, by way of size, charge, lipid solubility, water solubility or other characteristic, of moving from the lumen of a capillary to an interstitial fluid.

[0024]

[0021] , In one embodiment of the first aspect, the amount of the exogenous agent in the systemic circulation of the subject is determined by assay of the blood or a blood extract of the subject for the exogenous agent.

[0025]

[0022] , In one embodiment of the first aspect, the amount of the exogenous agent in the systemic circulation of the subject is predicted, inferred, or estimated by reference to (i) a known pharmacokinetic characteristic of the exogenous agent or a related exogenous agent, (ii) the amount of drug that is expected to enter into the systemic circulation of the subject after infusion, (iii) the amount of drug that is expected to enter the systemic circulation of the subject after oral ingestion, (iv) the amount of drug that is expected to enter the systemic circulation of the subject after intramuscular or subcutaneous injection, (v) population data, or (vi) theory.

[0026]

[0023] , In a second aspect, there is provided a method of assessing perfusion of a tissue in an animal or a human subject, the method comprising the steps of: administering an exogenous agent to the subject, at one or more time points after administration, determining an amount of the exogenous agent in the interstitial fluid of a tissue of the subject, wherein the determined amount is used on its own or in conjunction with another value to determine a tissue perfusion characteristic of the subject.

[0027]

[0024] , Embodiments of the second aspect may incorporate any one or more features of any embodiment of the first aspect.

[0028]

[0025] , In a third aspect, there is provided a method of titrating an exogenous agent so as to achieve a desired amount of the exogenous agent in a tissue of an animal or a human subject, the method comprising the steps of: administering the exogenous agent to the subject, at one or more time points after administration, determining an amount of the exogenous agent in the interstitial fluid of a tissue of the subject, wherein the determined amount is used on its own or in conjunction with another value to adjust the amount of the exogenous agent administered to the subject so as to achieve a desired amount of exogenous agent in the tissue.

[0029]

[0026] , Embodiments of the third aspect may incorporate any one or more features of any embodiment of the first aspect.

[0030]

[0027] , In a fourth aspect, there is provided a method for determining the tissue availability of an exogenous agent in the treatment or prophylaxis of a subject, the method comprising the steps of: administering the exogenous agent to the subject, at one or more time points after administration, determining an amount of the exogenous agent in the interstitial fluid of a tissue of the subject, wherein the determined amount is used on its own or in conjunction with another value to determine the tissue availability of the exogenous agent.

[0028] , Embodiments of the fourth aspect may incorporate any one or more features of any embodiment of the first aspect.

[0031]

[0029] , In a fifth aspect, there is provided a method of treating or preventing a condition in a subject, the method comprising administering to the subject an effective amount of a therapeutic or a prophylactic agent, wherein prior to the step of administering, identifying a tissue distribution phenotype according to the method of any embodiment of the first aspect, and determining an amount or an administration timing in relation to the therapeutic or a prophylactic agent by reference to the identified tissue distribution phenotype for the subject.

[0032]

[0030] , Embodiments of the fifth aspect may incorporate any one or more features of any embodiment of the first aspect

[0033] BRIEF DESCRIPTION OF THE FIGURES

[0034]

[0031] , FIG. 1 shows in graphical form the response of an EAB sensor specific to vancomycin (specifically a BASi Au electrode) in artificial ISF supplemented with protein.

[0035]

[0032] , FIG. 2 illustrates highly diagrammatically, a system configured to determine a distribution phenotype for a human subject.

[0036]

[0033] , FIG. 3 is a photograph showing a wearable EAB sensor as applied to the skin of the upper arm of a person.

[0037]

[0034] , FIG. 4A is a computer-rendered depiction of the surface of the EAB sensor used in the human studies described in the Example.

[0038]

[0035] , FIG. 4B is a diagrammatic cross-section of the EAB sensor of FIG. 4A.

[0039]

[0036] , FIG. 5 shows in graphical form the determination of maximum height of a plot of current versus potential.

[0040]

[0037] , FIG. 6 shows in graphical form a calibration plot of kinetic differential measurement (KDM) versus log vancomycin concentration.

[0041]

[0038] , FIG. 7 shows in graphical form the concentration of vancomycin in the serum and ISF of a single participant (identification code 012) of the human studies described in Example 3. Vancomycin concentration in the ISF was determined by an EAB sensor (identification code 812).

[0039] , FIG. 8 shows in graphical form the concentration of vancomycin in the serum and ISF of a single participant (identification code 001) of the human studies described in Example 3. Vancomycin concentration in the ISF was determined by an EAB sensor (identification code 816).

[0042]

[0040] , FIG. 9 shows in graphical form the concentration of vancomycin in the ISF of 6 subjects in the human studies described in Example 3.

[0043]

[0041] , FIG. 10 shows in graphical form the concentration of vancomycin in the ISF and serum (from blood sample) of subjects in the human studies described in Example 4.

[0044]

[0042] , FIG. 11 shows in graphical form the concentration of vancomycin in the ISF and serum (from blood sample) of subject in the human studies described in Example 5.

[0045]

[0043] , Unless otherwise indicated herein, features of the drawings labelled with the same numeral are taken to be the same features, or at least functionally similar features, when used across different drawings.

[0046]

[0044] , The drawings are not prepared to any particular scale or dimension and are not presented as being a completely accurate presentation of the various embodiments.

[0047]

[0045] , Unless otherwise indicated herein, features of the drawings labelled with the same numeral are taken to be the same features, or at least functionally similar features, when used across different drawings.

[0048]

[0046] , The drawings are not prepared to any particular scale or dimension and are not presented as being a completely accurate presentation of the various embodiments.

[0049] DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

[0050]

[0047] , After considering this description it will be apparent to one skilled in the art how the disclosure is implemented in various alternative embodiments and alternative applications. However, although various embodiments of the present disclosure will be described herein, it is understood that these embodiments are presented by way of example only, and not limitation. As such, this description of various alternative embodiments should not be construed to limit the scope or breadth of the present disclosure. Furthermore, statements of advantages or other aspects apply to specific exemplary embodiments, and not necessarily to all embodiments, or indeed any embodiment covered by the claims.

[0048] , Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

[0051]

[0049] , Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may.

[0052]

[0050] , The term “recognition element” includes any molecule(s) that, in the context of an electrochemical sensor, specifically interact with a target analyte of interest (such as a drug or a marker agent), the interaction causing a discernible change in the molecule(s). An analyte recognition element may be a polymer, and may comprise from about 5 to about 100 monomers, or from about 15 to about 50 monomers.

[0053]

[0051] , An aptamer is an exemplary form of analyte recognition element. Aptamers are small (usually from 20 to 60 nucleotides) RNA or DNA oligonucleotides formed from a single strand and able to bind a target analyte with high affinity and specificity. Aptamers may be considered as nucleotide analogues of antibodies, but aptamer production is an in vitro cell-free process that is significantly easier and cheaper than the production of antibodies by cell culture or in vivo methods. Aptamers typically comprise a polynucleotide sequence that promotes the assumption of 3 -dimensional shapes in the form of helices and single-stranded loops. Indeed, the specificity of aptamer binding is dictated not by the primary polynucleotide sequence, but instead by its 3 -dimensional structure, at least is part. In some circumstances, binding will be influenced by hydrophobic interactions, hydrogen bonding, Van der Waals forces, base-stacking, and intercalation.

[0054]

[0052] , An analyte recognition element may be a biological molecule or an analogue thereof. An exemplary analyte recognition element may be comprised of DNA, RNA, PNA, XNA. Single-stranded and double-stranded arrangements are contemplated.

[0055]

[0053] , An analyte recognition element may comprise a non-natural nucleic acid. As used herein, the term “non-natural nucleic acid” is intended to include a polymer that is biosimilar to a natural nucleic acid polymer such as DNA or RNA, but having a chemical structure that is altered and not found in nature. As a result of the altered structure, the nonnatural nucleic acid may be more resistant than a natural nucleic acid against degradation (such as cleavage of a chemical bond) occasioned by nucleases found in biological fluids such as blood and the ISF.

[0056]

[0054] , A non-natural nucleic acid may derive from a naturally occurring nucleic acid, but having had an alteration to its chemical structure such that the chemical structure is considered non-natural. More typically, the non-natural nucleic acid will be synthesised de novo in an altered form.

[0057]

[0055] , A non-natural nucleic acid molecule useful in the context of the present invention may be an altered form of an aptamer. The non-natural nucleic acid may be an oligomer having a non-natural backbone, being a molecular analogue to DNA or RNA. Examples of non-natural backbone oligomers include, but are not limited, to 2'-fluoroarabinoside nucleic acid (FANA), 2'-O-methyl RNA, locked nucleic acid (LNA), and threose nucleic acid (TNA). Collectively, these non-natural backbone oligomers are referred to as xeno nucleic acids (XNAs).

[0058]

[0056] , Apart from the altered chemical structure which confirms stability in biological fluids, a non-natural nucleic acids may share one or more general features of aptamers such as length, base sequence (primary structure), secondary structure and tertiary structure.

[0059]

[0057] , One method of identifying aptamers useful in the context of the present invention is to use a method of the prior art (such as SELEX) to identify a natural DNA or RNA aptamer, and optionally to then modify the identified aptamer so as to have a non-natural chemical structure. Alternatively, methods such as SELEX may be adapted by the use enzymes configured to synthesise and amplify non-natural nucleic acids in the first instance.

[0060]

[0058] , An analyte recognition element may be a protein. The protein may in the form of a peptide, optionally having a length of between 10 and 100 amino acids or longer. The protein may be in the form of a monomer, dimer, trimer, tetramer or higher. Antibodies, antibody fragments (such as Fab fragments) and antibody-like molecules may be useful, whether polyclonal or monoclonal.

[0061]

[0059] , As for polynucleotides, proteins may be subject to modification. For example, backbone modification may be used to improve proteolytic stability of the peptide. Backbone modification includes the substitution of L-amino acids by D-amino acids , insertion of methyl-amino acids, and the incorporation of P-amino acids and peptoids. Introducing these non-natural amino acids into the peptide sequence, particularly at a proteolysis site, is an effective strategy for improving resistance to proteases or other deleterious factors.

[0062]

[0060] . Side chain modifications may be achieved by replacing the natural amino acids with their analogues during peptide synthesis, to improve their binding affinity and target selectivity. Variants of natural amino acid analogues such as homoarginine, benzyloxytyrosine, and P-phenylalanine are commonly commercially available, and can be conveniently used to chemically modify the peptide side chain during peptide synthesis.

[0063]

[0061] , The weak forces in proteins, such as hydrogen bonds, van der Waals forces, and intramolecular hydrophobic interactions may not be adequate for a stable secondary structure conformation. Additional modifications of the backbone, N- or C-termini, or sidechains for stabilization of secondary structures may be pursued.

[0064]

[0062] , Cyclization is another potentially useful protein modification technique that can include various strategies, such as head-to-tail, backbone-to-side chain, and side chain-to- side chain cyclization. Cyclization can increase proteolytic stability, and allows mimicking and stabilization of the secondary structure.

[0065]

[0063] , The present disclosure is predicated at least in part on the inventors’ surprising discovery in the course of monitoring the amount of a drug in the interstitial fluid (ISF) of human subjects during a clinical trial of an electrochemical aptamer-based sensor. Particularly, it was found that the distribution of a drug from the circulation into the tissues varies to a large extent between different trial subjects.

[0066]

[0064] , As detailed in the Examples herein, the distribution of vancomycin into the tissues varies to the extent that one subject could be considered a “high distribution phenotype” and another subject considered a “low distribution phenotype”. Moreover, the kinetics of drug distribution have been found to differ between healthy subjects and subjects having a medical condition.

[0067]

[0065] , While it was known that the tissue-availability of a drug is influenced by subject variability such as the extent of liver metabolism, rates of renal clearance and the like, the present data shows that the subjects exhibited very similar pharmacokinetics (C max, Tmax, ti / 2) for vancomycin in the blood. It is therefore reasonable to rule out any inter-subject variation in tissue-availability due to individual variation in metabolism or renal clearance.

[0068]

[0066] , Without wishing to be limited by theory in any way, it is proposed that the data of the Examples shows differing extents to which a tissue is perfused between subjects, and also differing inter-subject kinetics. The differing levels of perfusion result in differing levels and rates of drug exiting the capillaries and entering the tissue beds. A more highly perfused or a more rapidly perfused tissue may be therefore exposed to higher amounts of drug, or higher maximum concentrations of drug. By contrast, drug will be sequestered away from the tissue (i.e. remain in the circulation) in tissue being perfused to a lesser extent. In addition or alternatively, drug may be sequestered away from tissue for a longer period of time.

[0069]

[0067] , Knowledge of a distribution phenotype of a subject may have utility in the clinical decision as to (i) the dosage of any drug used, or (ii) the dosing regime used, or (iii) even the drug used. For example, a subject having a “low” or a “slow” distribution phenotype for a drug may require higher dosages and / or more frequent dosing so as to achieve useful amounts of drug in the tissues. A subject having a “high” or a “fast” distribution phenotype may require lower dosages thereby limiting the potential for toxicity or drug wastage.

[0070]

[0068] , Where difficulty exists in achieving a desired tissue exposure, a different drug may be trialled. For example, a drug having a low water solubility may not be adequately transported along with the soluble components of plasma exiting the capillary. Testing the distribution of a more water soluble drug may reveal greater levels of distribution into the tissues, and in which case such a drug may present as a more suitable choice.

[0071]

[0069] , The unexpected differences in the extent and kinetics of drug perfusion may result in the need for calibration of a drug sensor such that the sensor output is clinically meaningful. A calibration method may be performed at first instance for each individual subject, and possibly multiple times for a given subject where the possibility exists that the subject’s tissue distribution phenotype has changed.

[0072]

[0070] , Calibration may be used to correlate a concentration of drug determined in the interstitial fluid (by sensor output) with a concentration in the central circulation, and without the need to assay blood. For example, it may be found in a calibration method that for a given concentration of a drug determined by a sensor in interstitial fluid is 50% of that assayed in the blood at the same time. Put another way, the drug may be found by way of calibration to partition in the tissues at a ratio of 1:2 (tissue concentration: blood concentration). Thus, establishing the partitioning ratio for a given subject allows for a sensor-determined drug concentration to estimate concentration in the blood.

[0073]

[0071] . The experimental work described herein demonstrates that EAB sensors presents a useful means by which the amount of a drug in the interstitial fluid may be monitored. Results may be provided in substantially real time, and in a manner that is minimally invasive. Moreover, the ability to continuously monitor the amount of a drug in the tissues over time may reveal a change in distribution. For example, a subject may be administered morphine for pain along with the drug under consideration, such as vancomycin. Morphine is known to induce histamine, which in turn can lead to an inflammatory response. Thus, an EAB sensor may detect an increase in vancomycin distribution to the tissues, and given the potential for toxicity the amount of vancomycin administered may be lowered.

[0074]

[0072] , The present disclosure may be exploited in a method for assessing the level of perfusion of a tissue. A drug required by the subject may be used to determine the extent of perfusion by a consideration of the amount that enters the interstitial fluid of the tissue. Alternatively, an inert market agent may be administered to the subject, with the amount of marker agent found in the tissues used as an indication of perfusion. Where an EAB sensor is used, the location of the sensor may be moved to different anatomical locations to assess local perfusion characteristics. A single subject may show a high level of perfusion in tissue of the arm, and low level in a tissue of the foot. Where an infection of the foot is the target of the drug, information on perfusion for that anatomical region particularly may be of significant clinical relevance.

[0075]

[0073] , In some instances, the level of perfusion may be of clinical interest for diagnosing a vascular disease, such as peripheral vascular disease, where demonstration of a low perfusion of a tissue may be supportive of a diagnosis. Alternatively, a high level of perfusion may be an indicator of an inflammatory process, or leaky capillary syndrome. For such applications, a marker agent rather than a drug may be administered to the subject, with the marker agent being detectable in the interstitial fluid of the tissue concerned. A suitable marker agent may be a generally inert molecule having a reasonable half-life in the circulation.

[0074] , The present disclosure may find further application is determining the suitability of a drug for a particular clinical indication based on the level to which the drug distributes to a target tissue. For example, two equally efficacious drugs may exist with the clinician being required to make a selection. One drug may be considered more suitable over the other on the basis of a higher level of distribution into the tissues as assessed by the methods disclosed herein.

[0076]

[0075] , A method for determining a tissue distribution phenotype for a subject in relation to a particular drug may be performed in combination with a method of prophylaxis or treatment of a condition for which that drug is efficacious and the subject is in need thereof. Thus, in the first part of the method a sensor is applied to the subject, and the drug or some type of marker agent is administered to the subject, and after a period of time or at a plurality of times, drug or marker agent concentration is determined contemporaneously by way of sensor data and blood assay, and a comparison is made between the sensor data and the assay data, and an amount and / or timing of the drug to be administered is determined by reference to the comparison. As a further step, the drug may be administered to the subject in accordance with the determined amount and / or timing.

[0077]

[0076] , The method may include the step of storage of comparative data or information, such as a multiple or a ratio or a correction factor, a lookup table, or a correlating factor for example. The data or information may be stored on the device itself, or in a remote location such as a hub, a server, a computer, or a mobile device such as a smartphone or a tablet.

[0078]

[0077] , In one aspect of the disclosure there may be provided an electrochemical sensor device or a sensor apparatus that electronically stores and / or transmits the comparative data or information to a remote device either by wired or wireless means.

[0079]

[0078] , Further utility may be found in a method for designing or screening a drug. A drug may be designed de novo or an existing drug may be modified so as to distribute effectively into the tissues. A candidate drug may be assessed in a human or a non-human animal by comparing levels of the drug in the interstitial fluid with levels in the systemic circulation. EXAMPLE 1: PRODUCTION OF VANCOMYCIN EAB SENSOR USING BASI AU ELECTRODE (d=1.6 mm)

[0080]

[0079] , Gold electrodes were electrochemically cleaned in 0.5 M NaOH, for cyclic voltammetry as follows:

[0081] Estart=-1.0 V; Eswitch=-1.6 V vs Ag|AgCl v = 1 V s'1

[0082] 200 cycles with Estep = 2 mV

[0083]

[0080] , Electrochemical treatment in 0.5 M H2SO4:

[0084]

[0081] , a) For platforms that do not require enhancement of sensing area (for cyclic voltammetry):

[0085] Estart = 0 V; Eswitch = +1 .6 V; Efmai= -0.2 V vs Ag|AgCl v = 100 mV s'1

[0086] 15 cycles

[0087] Estep 1 mV

[0088] Electrodes were washed 3 times with 1 mL of nuclease free FEO.

[0089]

[0082] , b) For platforms that requires enhancement of sensing area (for chronoamperometry): Ei = 0 V; E2= 2 V vs Ag|AgCl

[0090] Pulse length = 20 ms

[0091] Number of cycles = 16,000

[0092] Electrodes were washed 3 times with 1 mL of nuclease free H2O.

[0093]

[0083] , Electrode modification:

[0094] 2 pL of 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to

[0095] 2 pL of 100 pM DNA-aptamer for vancomycin [5ThioMC6- D / VancomycinDNA / 3MeBIN] and kept in the dark for 1 h. Solution is mixed with vortex.

[0096] Adjusted [5ThioMC6-D / VancomycinDNA / 3MeBIN] to 500 nM using PBS lx + 2 mM MgCl2. Solution was mixed with vortex.

[0097] All electrodes were kept immersed in the exact same solution for 1 hour in the dark. Electrodes were washed 3 times with 1 mL of PBS lx + 2 mM MgCl2.

[0098]

[0084] , All electrodes were incubated together in 20 mM 6-mercapto-l -hexanol in

[0099] PBS lx + 2 mM MgCl2for a minimum of 2 hours at room temperature and in the dark.

[0100]

[0085] , FIG. 1 shows in graphical form the response of a vancomycin sensing electrochemical drug sensor produced as described above in artificial ISF supplemented with protein.

[0101] EXAMPLE 2: SYSTEM FOR DETERMINING DRUG DIFFUSION PHENOTPYE OF A SUBJECT

[0102]

[0086] , Referring to FIG. 1, there is shown a system for monitoring the levels of a drug with a wearable device and administration of the drug. Particularly, the system comprises a wearable drug EAB sensor device (10) that is retained on the surface (15) of the skin of a subject (20) with an adhesive layer (not drawn) on the underside.

[0103]

[0087] , As will be noted from the cross-sectional magnified area inside the dashed square, the wearable device (10) comprises microneedles (one of which is marked (30)). The microneedles breach the stratum corneum which forms the skin surface (15) so as to contact the interstitial fluid of the underlying epidermis (35).

[0104]

[0088] , At least one of the microneedles (30) is configured as the working electrode of an EAB sensor, and is coated with an aptamer (not drawn) capable of selectively binding to the drug being monitored and administered. A counter electrode and a reference electrode is provided by further microneedle (30).

[0105]

[0089] , Each aptamer of the working electrode has an associated redox reporter configured such that the electrode outputs an electrical signal when drug binds to the aptamer. A power source (40) and circuitry (45) configured to provide electrical power are disposed within the wearable device (10). Also disposed within the wearable device are circuitry (50) configured to receive the signal output by the microneedles (30) and output in digital form to a wireless transmission module (55).

[0106]

[0090] , The system comprises a computer (60) having software (62) executed thereon for receiving data output by the wearable device (10).

[0107]

[0091] , Drug (held in reservoir (65)) is administered via the processor-controlled infusion pump (70), line (75) and cannula (80) to the circulation of the subject (20) via the venous route.

[0092] , From the circulation, the drug distributes throughout the body of the subject (20) and a proportion enters the interstitial fluid of the epidermis (35).

[0108]

[0093] , The drug binds to the aptamer-coated microneedles (30), and the resultant output signal processed by circuitry (50) and then passed to wireless communications module (55). The output signal (which is a drug concentration value) is transmitted wirelessly (85) to the computer (60).

[0109]

[0094] , In this system, samples of venous blood are taken from the subject (20) at intervals over the course of monitoring the amount of drug in interstitial fluid of the underlying epidermis (35). In a subject (20) exhibiting a high level of drug distribution from the circulation to the tissues, the concentration of drug in the interstitial fluid will approach that found in the circulation.

[0110] EXAMPLE 3: HIGH RESOLUTION VANCOMYCIN LEVEL DATA IN HEALTHY HUMAN SUBJECTSUSING AN EAB SENSOR CONTACTING INTERSTITIAL FLUID

[0111]

[0095] , These human studies followed vancomycin levels using an EAB sensor (which may also be referred to as the “device”). The device was a self-contained wearable EAB sensor, constructed, having an on-board power supply, electrodes, and electronics, including a microprocessor and BlueTooth™ communications module.

[0112]

[0096] , The EAB sensor comprised four electrodes: two aptamer-coated working electrodes, a counter electrode, and a reference electrode. All electrodes were in the form of a microneedle configured to pierce the skin and contact the participant’s ISF when fully inserted into the dermal tissues.

[0113]

[0097] , Each of the working electrodes were constructed from a gold plated acupuncture needle, cleaned by a plasma treatment before coating with aptamer in general accordance with the method described at Example 1.

[0114]

[0098] , Each working electrode was coated with a vancomycin-sensitive single-stranded DNA-aptamer having a length of 28 nucleobases, obtained from Dr. Milan STOJANOVIC, New York campus research laboratory of The Trustees of Columbia University in the City of New York of 80 Claremont Avenue, 4th Floor, New York, NY 10027, United States. The DNA-aptamer was previously demonstrated to specifically interact with vancomycin.

[0099] , Each entire device was disinfected by immersion for 10 minutes in Cidex™ (Johnson & Johnson) at room temperature. Devices were then rinsed in sterile phosphate buffered saline for 30 seconds.

[0115]

[0100] , The device comprised an adhesive disposed on a surface surrounding the electrodes, maintaining the device on the skin and therefore keeping the electrodes in contact with the interstitial fluid (see FIG. 3).

[0116]

[0101] , Reference is made to FIG. 4 A and FIG. 4B each showing a computer-rendered representation of the device (10) used in these studies, indicating one of the microneedles (30), the power supply, being a battery (105), a printed circuit board (110) having various electronic components such as the communications module and microprocessor mounted thereon, and a thermistor (115) functioning to contact the skin surface of the participant and provide an estimation of the temperature of the underlying ISF.

[0117]

[0102] , These human studies were performed at Monash Health, Monash Medical Centre,

[0118] Clayton, Victoria 3168, Australia, under protocol reference number 2021 / ETH80521 and protocol trial identified and registry 80521. Institutional Ethics Committee approval was obtained before commencement of the study.

[0119]

[0103] , Selection criteria used were as follows. Age 18-60 years. Individuals without clinically significant medical abnormalities contraindicating participation as determined by Study Investigators, including, but not limited to: (a) physical examination without any clinically relevant findings, (b) systolic blood pressure in the range of 90 to 140 mmHg (inclusive) and diastolic blood pressure in the range of 50 to 90 mmHg (inclusive) after 5 minutes of rest in a supine position, (c) pulse rate in the range of 60 to 100 bpm (inclusive) after 5 minutes of rest in a supine position. A 40-60 bpm (inclusive) may be considered acceptable for participants without clinically significant findings at the discretion of the Principal Investigator, (d) body temperature (tympanic), between 35.5°C and 37.5°C (inclusive), (e) no clinically significant findings in serum biochemistry, haematology tests, or urinalysis contraindicating participation as determined by Study Investigators.

[0120]

[0104] , Female participants of childbearing potential were required, from the period of signing the consent form until at least 28 days after the removal of the device: (a) to have a negative pregnancy test at screening and study visits, (b) not to be planning to become pregnant, (c) not to be breastfeeding, (d) not to donate ova. If engaging in sexual intercourse, they were required to use effective contraception during the studies and strongly recommended to use effective contraception for at least 28 days after the removal of the device.

[0121]

[0105] , Participation was not influenced by vaccination status. However, participants who had been vaccinated within one week of the study visit, were not recruited.

[0122]

[0106] , Exclusion criteria were as follows. Poor venous access for venepuncture. Participants who were currently receiving or have received any investigational drug / device within the last 30 days. History of allergic reactions to vancomycin, metals, plastics and adhesives which, in the opinion of the Study Investigators, would increase the risk of having allergic reactions associated with skin allergies, or vancomycin administration. Active illnesses. Consumption of prescription medications except oral contraceptive pills. Use of illicit drugs or alcohol consumption, which, in the opinion of the Study Investigators, may interfere with the completion of the studies.

[0123]

[0107] , A vancomycin-sensitive EAB sensor was placed onto the participant’s upper arm

[0124] (see FIG. 3), opposite the arm where vancomycin was being infused. The time of device application was recorded. The device was applied 30 minutes (+15 minutes) prior to administering the vancomycin infusion and the device was removed up to 10 hours postcessation of the infusion.

[0125]

[0108] , Blood samples for relevant pathology tests (FBC, UEC, and LFT) were collected (10 to 30 minutes prior to application of the device and 10 to 30 minutes post-removal of the device). If the participant consented to providing blood samples for future research, these were collected prior to the placement of the device, or once the device has been removed, at the time most convenient for the Clinical Team.

[0126]

[0109] , 30 minutes (+15 minutes) post-application of the device, the participant received a single dose of vancomycin as an intravenous infusion (1 gram over 1 hour 40 minutes). The time of administration and completion of infusion was recorded.

[0127]

[0110] , Blood samples for the measurement of vancomycin concentrations were collected prior to the administration of vancomycin infusion, 30 minutes (±5 minutes) and 1 hour (±10 minutes) during the infusion, at the end of the infusion (+15 minutes) and then at 40 minutes (±15 minutes), 1.5 hours (±15 minutes), 2 hours (±15 minutes), 3 hours (±15 minutes), 4 hours (±15 minutes), 6 hours (±15 minutes), 8 hours (±15 minutes) and 10 hours (±15 minutes) after the infusion has been completed.

[0128]

[0111] , Participants were required to complete a pain scale survey 5-10 minutes postapplication and 5-10 minutes after removal of the device.

[0129]

[0112] , Participants were required to conduct a physical challenge post-application of the device.

[0130]

[0113] , 5-10 minutes prior to removal of the device, a mobility survey was completed.

[0131]

[0114] , Digitally captured images / recordings of the skin surface at the device application site, before and after application and removal of the device, were taken to assess any skin irritation.

[0132]

[0115] , Participants were monitored throughout the study duration for any adverse events.

[0133] Given the duration of the study visit, participants were required to stay overnight. In the absence of any adverse events, participants were discharged the following morning after being observed for at least 15 minutes post removal of the device.

[0134]

[0116] , Application of the device for any reason and at any stage of the studies was as follows. Device application location was cleaned thoroughly with alcohol wipe (provided) as if the site were to be the site of an injection. The area was allowed to dry for 10 to 15 seconds before applying the device. An adhesive liner was removed from the bottom of the device being careful not to remove the safety tab. Prior to applying the device, the safety tab was still in the device. The device was applied to the cleaned site. Firm pressure was applied to the top of the device for 5 to 10 seconds. The device was applied with the safety tab pointing up. The safety tab was removed, and the top of the device was pressed such that the microneedles penetrated the skin. An audible click was heard as the device is pressed down, indicating that the microneedles were fully extended and locked in place.

[0135]

[0117] , Once applied to the participant, the DNA-based sensor electrodes were interrogated, and output treated as follows.

[0136]

[0118] , Square Wave Voltammetry (SWV) was used to interrogate the DNA-based sensor electrodes. Multiple steps were performed to convert the raw voltammograms obtained from the DNA-based sensor electrodes into a vancomycin concentration. The steps detailed below convert the raw SWV voltammograms into a signal that is indicative of the analyte concentration. 1. Smooth the raw SWV voltammogram current versus voltage data to aid identification of the current peak and its magnitude.

[0137] 2. Apply a peak finding algorithm to the smoothed voltammogram to identify the position of the peak and to subtract a baseline current to determine a peak current magnitude.

[0138] 3. Use the determined peak currents magnitudes obtained at two different SWV interrogation frequencies (in this case 50 Hz and 300 Hz), when no vancomycin is present and when it is present to calculate an analyte concentration responsive signal (S).

[0139] 4. Smooth the S values over time, prior to applying a calibration function.

[0140]

[0119] , Steps 1 to 3 were used to analyse the calibration data as detailed below at (a) and

[0141] (b), and steps 1 to 4 above in relation to the clinical data.

[0142] (a) Calibration data was produced from in vitro testing, where vancomycin is spiked into bovine plasma and tested using electrodes from the same production batch as those used in the clinical experiments (but not the electrode actually used in the clinical experiments). The purpose of this testing was to produce S versus known vancomycin concentration data that can be used to produce a calibration function to convert the S values into the corresponding measured vancomycin concentration values ([V]).

[0143] (b) Clinical experiment data, where the calibration function determined in (a) is applied to the S values produced by the in vivo electrodes in the clinical experiment, to convert them to measured vancomycin concentrations. This process produces [V] over time data.

[0144]

[0120] , The last step for the clinical data is to calculate the average [V] value of the two sensing electrodes (e. g. , working electrodes) in the same device at each time point to reduce random variation to give the final vancomycin concentration estimate.

[0145]

[0121] , Further detail in the process for generating vancomycin concentration as outline above will now be provided.

[0146] Voltammogram Smoothing

[0147]

[0122] , The first part of the peak-finding process is to smooth the measured current data.

[0148] This reduces noise and makes identification of peaks easier.

[0149]

[0123] , Smoothing was performed using a Savitzky-Golay filter. This filter moves along the array and fits a polynomial curve to a sliding window. Testing has shown that this filter performs well at eliminating noise but also retains peaks and troughs better than a rolling average approach. The raw data appears to tolerate aggressive filtering well thereby simplifying downstream peak-finding.

[0150] Baselining and peak measurement

[0151]

[0124] , The next step was to simply interpolate a baseline between the left and right troughs and identify the peak which is the maximum difference between the curve and the baseline when measuring vertically (and not perpendicular to the baseline).

[0152]

[0125] , The baselining approach uses the following process:

[0153] (i) Start at the left and right extremes of the curve.

[0154] (ii) Attempt to draw a straight line between the two points from the left to the right.

[0155] (iii) If at any point along this baseline, the actual curve is below the baseline then stop and move the left point one in along the curve.

[0156] (iv) Attempt to draw a straight line between the two points from the right to the left.

[0157] (v) If at any point along this baseline, the actual curve is below the baseline then stop and move the right point one in along the curve.

[0158] (vi) Repeat steps (ii) to (v) until either the two points meet (i.e., no peak was found - typically if the line is horizontal or straight) or until no portion of the curve is below the baseline (i.e., a valid peak was found).

[0159]

[0126] , Once a baseline had been identified, finding the peak was simply the point at which the difference between the curve and that baseline is maximized as shown in FIG. 22 xx5.

[0160]

[0127] , The following parameters can be used to modify the behavior of the peak-finding algorithm. The values used for the clinical data are given in the “Current Setting” column.

[0161] Producing analyte concentration responsive signal S

[0162]

[0128] , The peak current magnitude from four different voltammograms was combined to produce the S values. The four voltammograms were produced using two different SWV frequencies, interrogating two different solutions.

[0163]

[0129] , The two frequencies were chosen so that they respond differently to the concentration of vancomycin in the solution, with one frequency giving a larger increase in peak current as vancomycin concentration increases and one giving either a smaller increase or a decrease in peak current as the vancomycin concentration increases. The purpose of using the two frequencies is to aid in correcting for underlying drift in the peak current magnitude due to non-analyte related effects such as electrode fouling and loss of active aptamer from the surface of the electrode over time. In this case 300 Hz was chosen as the more strongly increasing frequency and 50 Hz as the less strongly increasing frequency.

[0164]

[0130] , The peak currents measured in the presence of vancomycin were divided by the peak currents measured for the same sensing electrode in the absence of vancomycin. This calculates a peak current signal gain caused by the presence of vancomycin and was used to cancel out variation between electrodes in the exact amount of analyte responsive aptamers present on the electrode.

[0165]

[0131] , The formula used to calculate S for an individual electrode in these studies is: where i300is the SWV peak current magnitude at 300 Hz in the test solution is the SWV peak current magnitude at 300 Hz in a solution with no vancomycin present i50is the SWV peak current magnitude at 50 Hz in the test solution iso is the SWV peak current magnitude at 50 Hz in a solution with no vancomycin present.

[0166]

[0132] , For the clinical data, the zero vancomycin peak current values used in the calculations were the values measured between 15 and 45 minutes after application of the device, when the signal had initially stabilised and before any vancomycin was infused into the participant.

[0167] Time Smoothing

[0168]

[0133] , The S values over time from the clinical data were then smoothed using a Sa iteky-

[0169] Golay. filter with a 41 point sliding window fitted to a second order polynomial, with the points taken at 5 minute intervals. The smoothed S values were used from this point on.

[0170] Producing the calibration function

[0171]

[0134] , To produce a calibration function that converts S values into [V] values, three electrodes from the same production batch of electrodes used in the clinical experiments were tested in bovine plasma containing a range of vancomycin concentrations at 35 °C. The plot shown at FIG. 6 is indicative of a calibration plot, where KDM corresponds to S values for multiple electrodes and the x-axis is the log of the known spiked concentration of vancomycin in the plasma.

[0172]

[0135] , Linear least squares was used to fit the S versus log(vancomycin concentration) data between 1 and 100 mg / L vancomycin concentration, to yield a straight line with Slope and an Intercept value. That is:

[0173] S = Slope. log([V]) + Intercept

[0174]

[0136] , The slope and intercept were used to convert S values from the clinical experiment into estimated vancomycin concentrations via the equation:

[0175]

[0137] , It will be noted that the approach detailed above is a departure from conventional means implementing a binding isotherm equation would be used. The above approach was used for simplicity.

[0176]

[0138] , Reference is made to FIG. 7 showing vancomycin concentrations measured in blood and skin ISF over time after an intravenous infusion of vancomycin. The concentration of vancomycin in ISF was determined by the EAB sensor described above. Blood expected peak is not measured. Blood peak or Cmax and trough is expressed at ~28 mg / L and ~4 mg / L, respectively. ISF peak or Cmax is ~6 mg / L. As will be noted, a marked difference between peak vancomycin concentration in blood (observed and expected) and ISF (observed) is noted, the latter being significantly lower). Total exposure (AUC) is also lower for ISF. Thus, in this subject the drug does not freely diffuse into the tissues, with the majority retained to a large extent in the circulation. The decrease in circulating amounts of vancomycin after the peak is presumably due to excretion (vancomycin not being appreciably metabolized by the liver). This subject may be considered to have “low” distribution phenotype for vancomycin, and having that knowledge the clinician may dose with higher amounts and / or at closer intervals to ensure the tissues are exposed to efficacious levels of the drug.

[0177]

[0139] , Reference is made to FIG. 8 showing vancomycin concentrations measured in blood and skin ISF over time after an intravenous infusion of vancomycin. Blood expected peak is not measured as blood dynamics are never expressed in the clinical setting. When blood peak is attempted based on timed draws it is obvious the peak is never measured but Cmax is observed and expressed. This subject (being a different individual to the subject of FIG. 7), could be considered to have a “high” distribution phenotype for vancomycin given that the peak drug concentration in ISF approaches the peak found in blood (observed or expected).

[0178]

[0140] , Reference is made to FIG. 9 showing vancomycin concentrations measured in skin

[0179] ISF over time after an intravenous infusion of vancomycin for multiple subjects in the human trial described above. Each subject exhibited different kinetics for the diffusion of vancomycin into the tissues about the EAB sensor. The rate and the extent of diffusion of the drug into the tissues was unique for each participant. These findings are consistent with the data of FIG. 7 and FIG. 8 which also showed considerable inter-subject variability

[0180]

[0141] , The distribution phenotypes for vancomycin as identified above may be considered also as an indicator of the level of perfusion of the tissue to which the EAB sensor is attached. Tissue may be perfused with fluid exiting the capillaries to different extents for different subject, but also for different tissues in the same subject. In that context, the type of exogenous agent administered to the subject may be a drug or a non-drug given that the aim of the exercise to determine the level of perfusion of a tissue. So long as the exogenous agent travels with the fluid exiting the capillaries and into the surrounding tissues, the extent to which the agent is found in the ISF is indicative of tissue perfusion.

[0181]

[0142] EXAMPLE 4: HIGH RESOLUTION VANCOMYCIN LEVEL DATA IN MULTIPLE HEALTHY HUMAN SUBJECTS USING AN EAB SENSOR CONTACTING INTERSTITIAL FLUID

[0182]

[0143] , This study was performed in general accordance with that described in Example 3 herein. Six healthy subjects were infused with vancomycin, with EAB sensor data being continuously recorded and blood samples taken for assay at regular intervals.

[0183]

[0144] , Reference is made to FIG. 10, showing a series of graphs of vancomycin concentration over a time period (subject #1 through subject #6). Each graph is directed to data obtained from a different subject. Each of the subjects received a vancomycin infusion over the period defined by “start” and “stop” on the graphs. The drug concentration graphs show to some extent that each of the six subjects responded generally in the same way in that tissue Cmax was lower, and Tmax later than that in the blood. Tissue also demonstrated a less defined peak drug concentration. Without wishing to be limited by theory in any way it is proposed that these comparative characteristics are indicative of some buffering of drug transport between the vascular compartment and the tissue compartment. Thus, the blood vessels may act as a depot for drug for a period of time, with drug moving into the tissue compartment by some rate-limited process. In a healthy individual fluid transfer across the blood vessel wall is controlled by the tight junctions formed between blood vessel endothelial cells of, these junctions possibly limiting the rate of fluid extravasation (and therefore drug) into the tissue.

[0184]

[0145] , The graphs typically show also a gradual decay in drug levels in the tissues, and more gradual as compared with the generally exponential decay in blood drug concentrations. The slower decay possibly indicates that drug tends to persist in the tissues, being relatively slowly drained into the lymphatic system over time. By contrast, blood is more rapidly depleted of drug presumably as a result of hepatic metabolism and renal clearance. These common features may allow for some assumptions to be made by a clinician in deciding a dosage regime, and without a need to consider blood assay data. For example, a drug may be dosed at a relatively high level so as to more quickly reach a therapeutic concentration in the tissues.

[0185]

[0146] , While the graphs of FIG. 10 show some consistent features, inter-subject variabilities are nevertheless evident. For example, drug appears to persist almost indefinitely in the tissue of subject #1. A clinician may elect to delay any further infusion of drug into a subject having a tissue distribution phenotype such as this, until tissue levels of the drug decline. It will be noted that the blood assay data for subject #1 fails to indicate the significant amount of drug persisting in the tissues.

[0186]

[0147] , In contrast to subject #1, drug clears from the tissues at a greater rate for subject

[0187] #2. For am individual having the tissue distribution phenotype of subject #2, the clinician may dose differently to subject #1. For subject #2, a second dosage may be given earlier given the more rapid decline in drug concentration from the tissues. An earlier dosage may prevent the drug form falling to a subtherapeutic concentration in the tissues.

[0188] EXAMPLE 5: INTERVENTIONAL STUDY - HIGH RESOLUTION VANCOMYCIN LEVEL DATA IN HUMAN SUBJECT IN INTENSIVE CARE UNIT USING AN EAB SENSOR CONTACTING INTERSTITIAL FLUID

[0189]

[0148] , This study was performed on a single subject according to registered clinical "Continuous monitoring of Intensive Care Unit (ICU) and Cardiac Care Unit (CCU) adult patients' vancomycin levels in interstitial fluid: a pilot characterization study of a new device", as published by the Australian New Zealand Clinical Trials Registry (w v^anzcb r^a ); ACTRN1 2625000208404.

[0190]

[0149] , Reference is made to FIG. 11, showing the periods in which the subject was infused with vancomycin (“start” to “stop”). Vancomycin concentration as assayed from blood is shown as the solid line. Vancomycin concentration as measured by EAB sensor is shown by light dots (first sensor device in place from 0 to 24h), and dark dots (second sensor device in place from 24h). As will be noted, tissue concentration of vancomycin more closely mirrors serum concentration, and especially after the second infusion. These data indicate a tissue distribution phenotype that is very different to the phenotypes of the healthy subjects of Example 4. Without wishing to be limited by theory in any way, many critically ill patients (and especially those with sepsis) have CLS (capillary leak syndrome) which manifests as derangement of fluid homeostasis between intravascular and extravascular spaces. Drug concentration curves for healthy subjects (having normal fluid homeostasis) as seen in Example 4 show a distinct buffering against a sharp Cmax peak because fluid (with drug) extravasates in a controlled manner and over time. For a patient with CLS, little control of extravasation is seen and drug is rapidly shunted into the tissues giving sharper peaks. Thus, the data obtained from the ICU patient in this Example 5 reveals a further tissue distribution phenotype. This CLS -type tissue distribution phenotype may suggest a specific drug dosing regime for a subject exhibiting that phenotype.

[0191]

[0150] , The data in FIG. 10 further demonstrates that a subj ect may change phenotype over time. After the first infusion of vancomycin, drug concentrations in the tissues plateaued after a rapid decay suggesting that a proportion of drug persisted in the tissues. That plateau was not noted after the second infusion. After the second and third infusion, drug did not appear to persist in the tissues with steady decay being noted until the next infusion was started. The change in phenotype may have been caused by administration of another drug, such as a diuretic, which causes loss of fluid from the tissues. Alternatively, the first infusion may have significantly cut bacterial load, thereby dampening the inflammatory process normally caused by bacterial infection under treatment. In any event, a clinician may use the change in tissue distribution phenotype to customize dosing the subject at a given time.

[0192]

[0151] , Those skilled in the art will appreciate that the disclosure is susceptible to further variations and modifications other than those specifically described. It is understood that the disclosure comprises all such variations and modifications which fall within the spirit and scope of the present disclosure.

[0193]

[0152] , Accordingly, the spirit and scope of the present disclosure is not to be limited by the foregoing examples, but is to be understood in the broadest sense allowable by law.

Claims

AMENDED CLAIMS received by the International Bureau on 03 September 2025 (03.09.2025)CLAIMS:

1. A method for determining a tissue distribution phenotype of an animal or a human subject for an exogenous agent, the method comprising the steps of: administering the exogenous agent to the subject, at one or more time points after administration, determining an amount of the exogenous agent in the interstitial fluid of a tissue of the subject, wherein the determined amount is used on its own or in conjunction with another value to determine a tissue distribution phenotype of the subject for the exogenous agent.

2. The method of claim 1, wherein the tissue distribution phenotype relates to a level to which, or a rate at which, the exogenous agent exits the capillaries and enters the interstitial fluid of a tissue of the subject.

3. The method of claim 1 or clam 2, wherein the tissue distribution phenotype is determined by reference to a concentration of the exogenous agent in blood of the subject, and a concentration of the exogenous agent in the interstitial fluid of a tissue of the subject.

4. The method of any one of clams 1 to 3, wherein the tissue distribution phenotype is determined by reference to a time series of concentrations of the exogenous agent in the interstitial fluid of the subject, optionally the time series being presented in graphical form.

5. The method of any one of claims 1 to 4, comprising the step of electronically storing comparative data or information relating to the determined amount of exogenous agent, and optionally the another value.

6. The method of any one of claims 1 to 5, comprising the steps of: applying to a tissue of the subject an electrochemical sensor based on a recognition element configured to specifically detect the exogenous agent such that a working electrode of the sensor contacts an interstitial fluid of the tissue, wherein the determination of an amount of the exogenous agent in the interstitial fluid is by made by reference to an output of the sensor.

7. The method of any one of claims 1 to 6, wherein the tissue distribution phenotype relates to the extent to which, or the rate at which, the exogenous agent distributes from the systemic circulation to the tissue.

8. The method of any one of claims 1 to 7, wherein the sensor comprises a working electrode that penetrates the skin so as to contact the interstitial fluid of the tissue.

9. The method of any one of claims 1 to 8, wherein the exogenous agent is a therapeutic agent or a prophylactic agent or a marker agent.

10. The method of any one of claims 7 to 9, wherein the amount of the exogenous agent in the systemic circulation of the subject is determined by assay of the blood or a blood extract of the subject for the exogenous agent.

11. The method of any one of claims 7 to 10, wherein the amount of the exogenous agent in the systemic circulation of the subject is predicted, inferred, or estimated by reference to (i) a known pharmacokinetic characteristic of the exogenous agent or a related exogenous agent, (ii) the amount of drug that is expected to enter into the systemic circulation of the subject after infusion, (iii) the amount of drug that is expected to enter the systemic circulation of the subject after oral ingestion, (iv) the amount of drug that is expected to enter the systemic circulation of the subject after intramuscular or subcutaneous injection, (v) population data, or (vi) theory.

12. A method of assessing perfusion of a tissue in an animal or a human subject, the method comprising the steps of: administering an exogenous agent to the subject, at one or more time points after administration, determining an amount of the exogenous agent in the interstitial fluid of a tissue of the subject, wherein the determined amount is used on its own or in conjunction with another value to determine a tissue perfusion characteristic of the subject.

13. The method of claim 12, comprising the steps of:applying to a tissue of the subject an electrochemical sensor based on a recognition element configured to specifically detect the exogenous agent such that a working electrode of the sensor contacts an interstitial fluid of the tissue, wherein the determination of an amount of the exogenous agent in the interstitial fluid is by made by reference to an output of the sensor.

14. The method of claim 12 or claim 13, wherein the determined amount is compared with an amount of the exogenous agent in the systemic circulation of the subject (i) determined contemporaneously or otherwise by assay, or (ii) predicted, inferred, or estimated.

15. The method of any one of claims 12 to 14, wherein the tissue perfusion characteristic relates to the extent to which, or the rate at which, the exogenous agent distributes from the systemic circulation to the tissue.

16. A method of titrating an exogenous agent so as to achieve a desired amount of the exogenous agent in a tissue of an animal or a human subject, the method comprising the steps of: determining a tissue distribution phenotype of the subject according to the method of any one of claims 1 to 15, administering the exogenous agent to the subject at a dosage based on the determined tissue distribution phenotype, at one or more time points after administration, determining an amount of the exogenous agent in the interstitial fluid of a tissue of the subject, wherein the determined amount is used on its own or in conjunction with another value to adjust the amount of the exogenous agent administered to the subject so as to achieve a desired amount of exogenous agent in the tissue.

17. The method of claim 16, comprising the steps of: applying to a tissue of the subject an electrochemical sensor based on a recognition element configured to specifically detect the exogenous agent such that a working electrode of the sensor contacts an interstitial fluid of the tissue, wherein the determination of an amount of the exogenous agent in the interstitial fluid is by made by reference to an output of the sensor.

18. The method of claim 16 or claim 17, wherein the determined amount is compared with an amount of the exogenous agent in the systemic circulation of the subject (i) determined contemporaneously or otherwise by assay, or (ii) predicted, inferred, or estimated.

19. A method for determining the tissue availability of an exogenous agent in the treatment or prophylaxis of a subject, the method comprising the steps of: determining a tissue distribution phenotype of the subject according to the method of any one of claims 1 to 15, administering the exogenous agent to the subject at a dosage based on the determined tissue distribution phenotype, at one or more time points after administration, determining an amount of the exogenous agent in the interstitial fluid of a tissue of the subject, wherein the determined amount is used on its own or in conjunction with another value to determine the tissue availability of the exogenous agent.

20. The method of claim 19, comprising the steps of: applying to a tissue of the subject an electrochemical sensor based on a recognition element configured to specifically detect the exogenous agent such that a working electrode of the sensor contacts an interstitial fluid of the tissue, wherein the determination of an amount of the exogenous agent in the interstitial fluid is by made by reference to an output of the sensor.

21. The method of claim 19 or claim 20, wherein the determined amount is compared with an amount of the exogenous agent in the systemic circulation of the subject (i) determined contemporaneously or otherwise by assay, or (ii) predicted, inferred, or estimated.

22. The method of any one of claims 19 to 21, wherein the tissue availability relates to the extent to which, or the rate at which, the exogenous agent distributes from the systemic circulation to the tissue.

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